Colloids with nanoporous structures and devices and systems for non-enzymatic glucose sensing

Abstract

The present invention is entitled "Colloid with Nanoporous Structure and Device and System for Non-enzymatic Glucose Sensing." This disclosure relates to a colloidal composition comprising a plurality of nanoparticle clusters dispersed in a liquid, a nanoporous layer formed from said colloid, a glucose oxidation electrode including said nanoporous layer, and a glucose sensing device, apparatus, and system including said glucose oxidation electrode. This disclosure also relates to a method for preparing said colloidal composition, said nanoporous layer, said glucose oxidation electrode, and said glucose sensing device and system. Furthermore, this disclosure relates to devices, systems, and methods for continuous glucose monitoring (CGM) and blood glucose monitoring (BGM).

Description

[0001] This application is a divisional application of the Chinese national phase patent application No. 201880073713.7, which was filed on May 14, 2020, after the international application No. PCT / US2018 / 065838, the international application date was December 14, 2018, and the invention was entitled "Colloid with Nanoporous Structure and Device and System for Non-enzymatic Glucose Sensing". Background Technology Technical Field

[0003] This disclosure relates to glucose sensing.

[0004] Discussion of related technologies

[0005] In the healthcare industry, there is great interest in technologies that improve the sensing and monitoring of blood glucose levels. Currently, most glucose sensors use electrochemical methods. Most (if not all) electrochemical sensors use enzyme-based electrochemical sensors. Summary of the Invention

[0006] One aspect of the present invention provides a colloidal composition comprising: a plurality of nanoparticle clusters dispersed in a liquid, wherein each cluster comprises a plurality of nanoparticles that aggregate together to form an irregularly shaped body having a nanometer or micrometer length, wherein the individual nanoparticles have a generally elliptical or spherical discrete body with a diameter of about 2 nm to about 5 nm, wherein interparticle gaps are formed between adjacent nanoparticles within each cluster and have an interparticle gap distance of about 0.5 nm to about 2 nm.

[0007] In the above colloidal composition, interparticle spaces are typically distributed throughout the clusters. The composition may be substantially surfactant-free. The liquid may comprise water, and the colloidal composition may contain less than 2 parts by weight of surfactant per 100 parts by weight of the nanoparticles contained therein. The amount of nanoparticles contained in the colloidal composition, based on the total weight of the colloidal composition, may be between about 0.01 wt% and about 2 wt%. The amount of nanoparticles contained in the colloidal composition, based on the total weight of the colloidal composition, may be between about 0.01 wt% and about 1 wt%.

[0008] In the aforementioned colloidal composition, the nanoparticles are primarily made of at least one selected from the group consisting of: platinum (Pt), gold (Au), palladium (Pd), rhodium (Rh), titanium (Ti), ruthenium (Ru), tin (Sn), nickel (Ni), copper (Cu), indium (In), thallium (Tl), zirconium (Zr), iridium (Ir), and one or more oxides of each of the above elements. The nanoparticles are primarily made of platinum (Pt), wherein the interparticle spaces are typically distributed throughout the clusters. The colloidal composition may contain less than 1 part by weight of surfactant per 100 parts by weight of the nanoparticles contained therein, wherein the amount of nanoparticles contained in the colloidal composition, based on the total weight of the colloidal composition, is between about 0.1 wt% and about 1 wt%.

[0009] Another aspect of the present invention provides a method for preparing a nanoporous layer. The method includes: dispensing the above-described colloidal composition onto a substrate; subjecting the dispensed colloidal composition to drying such that clusters contained in the dispensed composition are deposited on the substrate and stacked on top of each other to provide a nanoporous layer on the substrate, wherein the nanoporous layer comprises an irregularly shaped body formed by the stacked clusters, wherein the irregularly shaped body comprises a plurality of nanoparticles locally aggregated and interparticle gaps formed between adjacent nanoparticles within the irregularly shaped body, wherein the irregularly shaped bodies are interconnected to provide a three-dimensional interconnected network of irregularly shaped bodies, wherein irregularly shaped spaces are formed between adjacent portions of the irregularly shaped bodies and are nanoscale or microscale in size.

[0010] In the above method, the nanoparticles may be generally elliptical or spherical, with a diameter of about 2 nm to about 5 nm. The interparticle spacing may have an interparticle distance of about 0.5 nm to about 2 nm. Irregularly shaped spaces may be interconnected to provide a three-dimensional interconnected network of irregularly shaped spaces. The colloidal composition may be dispensed in a predetermined amount to form a nanoporous layer having a roughness coefficient between about 100 and about 2500. The nanoporous layer may contain less than 0.5 parts by weight of surfactant per 100 parts by weight of the nanoparticles contained therein.

[0011] Another aspect of the present invention provides a method for preparing a colloidal composition. The method includes: providing a liquid composition comprising metal ions, a surfactant, and a solvent, wherein the surfactant is situated in a reverse micelle phase defining a hydrophilic space; adding a reducing agent to the liquid composition to reduce the metal ions, thereby forming a first colloid comprising metal nanoparticles and a surfactant, wherein the metal nanoparticles are dispersed together with the reverse micelle phase of the surfactant in the first colloid; and removing the surfactant from the first colloid to provide a second colloid comprising a plurality of clusters dispersed in the liquid, wherein each cluster comprises a plurality of nanoparticles aggregated together to form an irregularly shaped body having a nanometer or micrometer length.

[0012] In the above preparation method, no potential needs to be applied to the liquid composition for the reduction of metal ions therein. The surfactant can be a nonionic surfactant capable of forming an isotropic reverse micelle phase. Individual nanoparticles can have a generally elliptical or spherical discrete bulk with a diameter of about 2 nm to about 5 nm, wherein interparticle gaps can be formed between adjacent nanoparticles within each cluster and have an interparticle gap distance of about 0.5 nm to about 2 nm. Surfactant removal involves removing a significant amount of surfactant from the first colloid so that the second colloid is substantially surfactant-free. Further surfactant removal involves removing a significant amount of surfactant from the first colloid so that the second colloid contains less than 1 part by weight of surfactant per 100 parts by weight of the nanoparticles contained therein.

[0013] In the above preparation method, removing the surfactant may include: centrifuging the first colloid; and collecting the bottom portion from the centrifuged composition. Removing the surfactant may also include repeatedly performing a series of centrifugations and collections. Removing the surfactant may also include adding an acid or base to the first colloid prior to centrifugation. Removing the surfactant may also include repeatedly performing a series of the following operations: addition, centrifugation, and collection. The amount of nanoparticles contained in the second colloid, based on the total weight of the composition, may be between about 10 wt% and about 40 wt%. The nanoparticles may be primarily made from at least one selected from the group consisting of: platinum (Pt), gold (Au), palladium (Pd), rhodium (Rh), titanium (Ti), ruthenium (Ru), tin (Sn), nickel (Ni), copper (Cu), indium (In), thallium (Tl), zirconium (Zr), iridium (Ir), and one or more oxides of each of the above metals. The nanoparticles are mainly made of platinum (Pt), and the interparticle spaces are usually distributed throughout the clusters. The composition may contain less than 2 parts by weight of surfactant per 100 parts by weight of the nanoparticles contained therein. The amount of nanoparticles contained in the composition, based on the total weight of the composition, may be between about 0.1 wt% and about 2 wt%.

[0014] Another aspect of the present invention provides a method for preparing a nanoporous layer. This method includes the above-described method for preparing a colloidal composition to provide a second colloid; dispensing the second colloid onto a substrate; subjecting the dispensed second colloid to drying such that clusters contained in the dispensed composition are deposited on the substrate and further stacked on top of each other to provide a nanoporous layer on the substrate, wherein the nanoporous layer comprises an irregularly shaped body formed by the stacked clusters, wherein the irregularly shaped body comprises a plurality of nanoparticles locally aggregated and interparticle gaps formed between adjacent nanoparticles within the irregularly shaped body. The irregularly shaped bodies are interconnected to provide a three-dimensional interconnection network of irregularly shaped bodies, wherein irregularly shaped spaces are formed between adjacent portions of the irregularly shaped bodies and are nanoscale or microscale in size, wherein the irregularly shaped spaces are interconnected to provide a three-dimensional interconnection network of irregularly shaped spaces.

[0015] In the above-described method for manufacturing a nanoporous layer, the nanoparticles may be generally elliptical or spherical, having a diameter of about 2 nm to about 5 nm, wherein the interparticle spacing has an interparticle distance of about 0.5 nm to about 2 nm. The colloidal composition may be dispensed in a predetermined amount to form a nanoporous layer having a roughness coefficient between about 100 and about 2500. The nanoporous layer may contain less than 0.1 parts by weight of a surfactant per 100 parts by weight of the nanoparticles contained therein.

[0016] Another aspect of the present invention provides a nanoporous structure comprising: an irregularly shaped body including a plurality of nanoparticles locally aggregated together and interparticle gaps formed between adjacent nanoparticles in the irregularly shaped body, wherein the nanoparticles may be generally elliptical or spherical and have a diameter of about 2 nm to about 5 nm, wherein the interparticle gaps have an interparticle gap distance of about 0.5 nm to about 2 nm, wherein the irregularly shaped bodies may be interconnected to provide a three-dimensional interconnection network of the irregularly shaped bodies, wherein irregularly shaped spaces are formed between adjacent portions of the irregularly shaped bodies and are nanoscale or microscale, wherein the irregularly shaped spaces are interconnected to provide a three-dimensional interconnection network of the irregularly shaped spaces.

[0017] The aforementioned nanoporous structures are generally free of surfactant molecules. In these nanoporous structures, the interparticle gaps are generally free of nanoscale organic molecules. The three-dimensional network of the irregularly shaped bulk and the three-dimensional network of the inter-cluster gaps are complementary to form the nanoporous structure. The interparticle gaps are generally self-interconnected and can also connect to the three-dimensional interconnection network of the inter-cluster gaps. The nanoporous structure can be formed by distributing a solid-liquid colloid containing irregularly shaped discrete clusters dispersed in a liquid and drying the distributed solid-liquid colloid, wherein the irregularly shaped discrete clusters can be stacked to provide a three-dimensional interconnection network of the irregularly shaped bulk and a three-dimensional interconnection network of the inter-cluster gaps. The inter-cluster gaps have an average inter-cluster gap distance. Nanoparticles can be made from at least one of the following: platinum (Pt), gold (Au), palladium (Pd), rhodium (Rh), titanium (Ti), ruthenium (Ru), tin (Sn), nickel (Ni), copper (Cu), indium (In), thallium (Tl), zirconium (Zr), iridium (Ir), and one or more oxides of each of the above metals. The nanoporous structure has a roughness coefficient between about 100 and about 2500.

[0018] Another aspect of the present invention provides an apparatus comprising: a substrate including a surface; and a nanoporous layer formed on the surface and including the aforementioned nanoporous structure. Yet another aspect of the present invention provides a non-enzymatic glucose sensing electrode comprising: at least one conductive layer including a surface; and a nanoporous layer formed on the surface and including the aforementioned nanoporous structure, wherein the non-enzymatic glucose sensing electrode does not contain a glucose-specific enzyme.

[0019] In the aforementioned device or electrode, at least one conductive layer may include a conductive metal layer and a conductive carbon layer formed on the conductive metal layer. The device or electrode does not include a biocompatible polymeric material formed on a nanoporous layer. The device or electrode may include a biocompatible polymeric material formed on a nanoporous layer.

[0020] In another aspect, the present invention provides a single-use glucose sensing device comprising: a reservoir configured to receive and contain a test liquid; and the aforementioned electrode arranged together with the reservoir such that a nanoporous layer can contact the test liquid when the test liquid is contained in the reservoir. In the single-use glucose sensing device, the electrode does not include a biocompatible polymeric material formed on the nanoporous layer.

[0021] In another aspect, the present invention provides a continuous glucose monitoring (CGM) device comprising: a subcutaneous injection needle configured to contact tissue fluid in a subject's body; and circuitry connected to the subcutaneous injection needle, wherein the subcutaneous injection needle includes the aforementioned electrode and another electrode connected to the circuitry.

[0022] In another aspect, the present invention provides a non-enzymatic glucose sensing device comprising: a working electrode including a substrate and a nanoporous layer formed on the substrate, the working electrode not containing a glucose-specific enzyme, wherein the nanoporous layer may include irregularly shaped bodies comprising a plurality of nanoparticles locally aggregated together, wherein interparticle gaps may be formed between adjacent nanoparticles of the irregularly shaped bodies, wherein the nanoparticles may be generally elliptical or spherical and have a diameter of about 2 nm to about 5 nm, wherein the interparticle gaps have an interparticle gap distance of about 0.5 nm to about 2 nm, wherein the irregularly shaped bodies may be interconnected to provide a three-dimensional interconnection network of irregularly shaped bodies, which generally extends throughout the nanoporous layer, wherein irregularly shaped spaces may be formed between adjacent portions of the irregularly shaped bodies and may be nanoscale or microscale, wherein the irregularly shaped spaces may be interconnected to provide a three-dimensional interconnection network of irregularly shaped spaces, which generally extends throughout the nanoporous layer, wherein the nanoporous layer may be configured to oxidize glucose molecules in the absence of a glucose-specific enzyme when a bias voltage between about 0.2 V and about 0.45 V is applied thereto.

[0023] In the aforementioned non-enzymatic glucose sensing device, the nanoporous layer is generally free of surfactant molecules, and the substrate may include at least one conductive layer comprising a conductive or semi-conductive material. The interparticle gaps are generally free of nanoscale organic molecules. The three-dimensional network of irregularly shaped bulk particles and the three-dimensional network of inter-cluster gaps are complementary to form the nanoporous layer. The interparticle gaps are generally self-interconnected and can also connect to the three-dimensional interconnection network of inter-cluster gaps.

[0024] In the aforementioned non-enzymatic glucose sensing device, the nanoporous layer can be formed by distributing a solid-liquid colloid comprising irregularly shaped discrete clusters dispersed in a liquid and drying the distributed solid-liquid colloid. The irregularly shaped discrete clusters can be stacked to provide a three-dimensional interconnect network of irregularly shaped bulks and a three-dimensional interconnect network of gaps between the irregularly shaped clusters. The nanoparticles can be made of at least one selected from the group consisting of platinum (Pt), gold (Au), palladium (Pd), rhodium (Rh), titanium (Ti), ruthenium (Ru), tin (Sn), nickel (Ni), copper (Cu), indium (In), thallium (Tl), zirconium (Zr), iridium (Ir), and one or more oxides of each of the aforementioned metals. The nanoporous layer has a roughness coefficient between about 100 and about 2500. The nanoporous electrode may also include a maltose barrier layer formed on the nanoporous layer and configured to substantially block maltose contained in the test fluid from passing through it, while allowing glucose to pass through. The maltose barrier layer may contain polyphenylene diamine (poly-PD), whose morphology allows glucose molecules to pass through while effectively blocking maltose molecules from passing through. The bias voltage can be set in the range of 0.2V to 0.45V.

[0025] In another aspect, the present invention provides a non-enzymatic glucose sensing system, comprising: the aforementioned non-enzymatic glucose sensing device; a counter electrode; and a bias voltage supply electrically connected between the working electrode and the counter electrode for supplying a bias voltage between the working electrode and the counter electrode.

[0026] In another aspect, the present invention provides a non-enzymatic glucose sensing method. The method includes: providing the aforementioned non-enzymatic glucose sensing device; applying a bias voltage between the working electrode and the counter electrode when a test fluid contacts both a working electrode and a counter electrode, causing glucose contained in the test fluid to oxidize in a nanoporous layer; measuring a current from the working electrode; and processing the current, with or without additional data, to provide a glucose level corresponding to the glucose contained in the test fluid. The bias voltage can be set in the range between 0.2V and 0.45V.

[0027] Another aspect of the present invention provides a glucose sensing electrode comprising: a substrate; a nanoporous metal layer formed on the substrate and capable of oxidizing both glucose and maltose in the absence of enzymes specific to glucose or maltose in the glucose sensing electrode; and a maltose barrier layer formed on the nanoporous metal layer. In the glucose sensing electrode, the maltose barrier layer has porosity that allows glucose to pass through and inhibits maltose from passing through the nanoporous metal layer, such that when a bias voltage of 0.2-0.45V is applied to the nanoporous metal layer relative to a reference electrode and when the maltose barrier layer is in contact with a liquid containing 4-20mM glucose and 4-20mM maltose, the current generated solely by glucose oxidation in the nanoporous metal layer is greater than 10 nA / mMcm. 2 Furthermore, it also reduces the current generated solely by the oxidation of maltose in the nanoporous metal layer to below 5 nA / mMcm. 2 .

[0028] In the aforementioned glucose sensing electrode, the nanoporous metal layer is capable of oxidizing glucose, such that when a bias voltage of 0.2-0.45V is applied and the electrode is in contact with a liquid containing 4-20 mM glucose without a maltose barrier layer, the current generated solely by glucose oxidation exceeds 10 nA / mMcm. 2 The nanoporous metal layer can also oxidize maltose, such that when a bias voltage of 0.2-0.45V is applied and the liquid contains 4-20mM maltose in contact with it without a maltose barrier layer, the current generated solely by maltose oxidation exceeds 10 nA / mMcm. 2 The maltose barrier layer may contain polyphenylene diamine (poly-PD) and have a thickness between 10 nm and 40 nm. The maltose barrier layer may essentially be composed of polyphenylene diamine (poly-PD) and have a thickness between 10 nm and 35 nm. The maltose barrier layer may be composed of polyphenylene diamine (poly-PD) and have a thickness between 10 nm and 40 nm.

[0029] In the aforementioned glucose sensing electrode, the nanoporous metal layer may include an irregularly shaped substrate comprising a plurality of locally aggregated nanoparticles and interparticle gaps formed between adjacent nanoparticles within the irregularly shaped substrate. Here, the nanoparticles are generally elliptical or spherical, having a diameter of approximately 2 nm to approximately 5 nm. The interparticle gaps may have a spacing of approximately 0.5 nm to approximately 2 nm. The irregularly shaped substrates may be interconnected to provide a three-dimensional interconnection network. Irregularly shaped spaces may be formed between adjacent portions of the irregularly shaped substrates and are nanoscale or microscale in size. The irregularly shaped spaces may be interconnected to provide a three-dimensional interconnection network.

[0030] The glucose sensing electrode described above may further include an electrolyte ion blocking layer formed on the maltose blocking layer and a biocompatible layer formed on the electrolyte ion blocking layer. The electrolyte ion blocking layer is configured to suppress Na+ contained in the liquid. + K + Ca 2+ Cl - PO4 3- and CO3 2- Diffusion into the nanoporous metal layer, so that Na exists between the top and bottom of the electrolyte ion barrier layer. + K + Ca 2+ Cl - PO4 3- and CO3 2- The combined concentrations exhibit a general discontinuity. The electrolyte ion barrier layer facilitates the modulation of the glucose sensing electrode, enabling modulation to be achieved within 30 minutes of contact with the subject's body fluids by applying a bias voltage of 0.2–0.45 V.

[0031] Another aspect of the present invention provides a device comprising: a single integrated body including a subcutaneous portion and a terminal portion; the subcutaneous portion including the aforementioned glucose sensing electrode and reference electrode, wherein when the subcutaneous portion is subcutaneously inserted into the body of a first subject, the two electrodes are each exposed to contact the tissue fluid of the first subject; and the terminal portion is configured to couple with a corresponding device and includes a first terminal electrically connected to the glucose sensing electrode and a second terminal electrically connected to the reference electrode.

[0032] In another aspect, the present invention provides an apparatus comprising: a single integrated body including the aforementioned glucose sensing electrode and reference electrode, the single integrated body further comprising a reservoir configured to at least temporarily contain a test fluid therein, wherein the glucose sensing electrode and the reference electrode are arranged in the single integrated body such that when the test fluid is contained in the reservoir, the glucose sensing electrode and the reference electrode are each configured to contact the test fluid.

[0033] Another aspect of the present invention provides a method for manufacturing a glucose sensing electrode. The method includes: providing a nanoporous metal layer capable of oxidizing both glucose and maltose in the absence of enzymes specific to glucose or maltose in the glucose sensing electrode; and forming a polyphenylene diamine (poly-PD) film on the nanoporous platinum layer such that the poly-PD film allows glucose to pass through while blocking maltose from passing through. Here, the poly-PD film has porosity that allows glucose to pass through while inhibiting maltose from passing through the nanoporous metal layer, such that when a bias voltage of 0.2-0.45V is applied to the nanoporous metal layer relative to a reference electrode and when the poly-PD film is in contact with a liquid containing 4-20 mM glucose and 4-20 mM maltose, the current generated solely by glucose oxidation in the nanoporous metal layer is greater than 10 nA / mMcm. 2 Furthermore, it also reduces the current generated solely by the oxidation of maltose in the nanoporous metal layer to below 5 nA / mMcm. 2 .

[0034] In the above-described method for manufacturing a glucose sensing electrode, forming a poly-PD thin film may include using a nanoporous metal layer as an electrode for electrochemical polymerization. Forming the poly-PD thin film may include providing a polymer layer containing poly-PD and adjusting the porosity of the polymer layer when it lacks sufficient porosity to allow glucose to pass through, such that the current generated solely by glucose oxidation in the nanoporous metal layer is less than 10 nA / mMcm. 2 Adjusting the porosity may include subjecting the polymer layer to at least one electric shock when it comes into contact with an acidic solution. Forming a poly-PD film may include polymerizing poly-PD from a liquid composition containing a certain concentration of phenylenediamine, wherein when the concentration is higher than a predetermined value, forming the poly-PD film further includes adjusting the porosity of the polymer layer. Adjusting the porosity may include subjecting the polymer layer to at least one electric shock when it comes into contact with an acidic solution.

[0035] In the above-described method for manufacturing a glucose sensing electrode, forming a poly-PD thin film may include providing a polymer layer comprising poly-PD, without further adjusting the porosity of the polymer layer to allow glucose to pass through it, such that the current generated solely by glucose oxidation in the nanoporous metal layer is expected to be higher than 10 nA / mMcm. 2 Forming a poly-PD film may include polymerizing poly-PD from a liquid composition containing a certain concentration of phenylenediamine, wherein when the concentration is below a predetermined value, the method does not include adjusting the porosity of the polymer layer to form a poly-PD film.

[0036] One aspect of the present invention provides a glucose sensing electrode, comprising: a conductive layer; a nanoporous metal layer formed on the conductive layer; an electrolyte ion blocking layer formed on the nanoporous metal layer; and a biocompatible layer formed on the electrolyte ion blocking layer. The glucose sensing electrode does not include a glucose-specific enzyme. When in contact with substances containing glucose and sodium... + K + Ca 2+ Cl - PO4 3- and CO3 2- In liquids containing sodium ions, an electrolyte ion barrier layer is configured to suppress sodium ions in the liquid. + K + Ca 2 + Cl - PO4 3- and CO3 2- Diffusion into the nanoporous metal layer, so that Na exists between the top and bottom of the electrolyte ion barrier layer. + K + Ca 2+ Cl - PO4 3- and CO3 2- The general discontinuity of the combined concentration.

[0037] In the aforementioned glucose sensing electrode, when a bias voltage of 0.2-0.45V is applied relative to a reference electrode, the glucose sensing electrode is configured to oxidize glucose in the nanoporous metal layer and generate a current. This current is the sum of the glucose oxidation current generated solely by glucose oxidation and the background current generated by other electrochemical interactions between the liquid and the glucose sensing electrode. When the liquid contains a glucose concentration of 4-20 mM (approximately 72-360 mg / dL), the glucose oxidation current is above 10 nA / mMcm under steady-state conditions. 2 At the level of.

[0038] In the aforementioned glucose sensing electrode, the combined concentration below the electrolyte ion barrier layer is greater than 0% and less than about 10% of the combined concentration above the electrolyte ion barrier layer. The combined concentration below the electrolyte ion barrier layer is greater than 0% and less than about 5% of the combined concentration above the electrolyte ion barrier layer. The electrolyte ion barrier layer may comprise a porous hydrophobic polymer layer configured to confine Na+. + K + Ca 2+ Cl - PO4 3- and CO3 2-The molecules migrate through it without restricting the migration of glucose molecules through it.

[0039] In the glucose sensing electrode described above, the electrolyte ion blocking layer may include at least one selected from the group consisting of: poly(methyl methacrylate) (PMMA), poly(hydroxyethyl methacrylate) (PHEMA), and poly(methyl methacrylate-co-ethylene glycol dimethacrylate) (PMMA-EG-PMMA). The electrolyte ion blocking layer may comprise at least one selected from the group consisting of: a copolymer of methyl methacrylate and butyl methacrylate; and a polymer obtained by polymerization of one or more monomers, said one or more monomers including branched or unbranched C1-C8 alkyl methacrylate, branched or unbranched C1-C8 cycloalkyl methacrylate, branched or unbranched C1-C8 alkyl acrylate, branched or unbranched C1-C8 cycloalkyl acrylate, and branched or unbranched C1-C8 cycloalkyl methacrylate, wherein said one or more monomers are selected from the group consisting of: methyl methacrylate, ethyl methacrylate, propyl methacrylate, butyl methacrylate, pentyl methacrylate, hexyl methacrylate, cyclohexyl methacrylate, 2-ethylhexyl methacrylate, methyl acrylate, ethyl acrylate, propyl acrylate, butyl acrylate, pentyl acrylate, hexyl acrylate, cyclohexyl acrylate, and 2-ethylhexyl acrylate.

[0040] In the aforementioned glucose sensing electrode, the glucose sensing electrode can be a continuous glucose monitoring (CGM) electrode, wherein the liquid is the subject's body fluid. An electrolyte ion-blocking layer is configured to facilitate the modulation of the glucose sensing electrode, such that modulation is accomplished within 30 minutes of contact with the subject's body fluid by applying a bias voltage of 0.2-0.45V. Modulation of the glucose sensing electrode is considered complete when the current decay rate is less than a first predetermined value and / or when the current remains less than a second predetermined value.

[0041] The glucose sensing electrode may further include a maltose barrier layer inserted between a nanoporous metal layer and an electrolyte ion barrier layer, wherein the maltose barrier layer may comprise polyphenylene diamine (poly-PD). The maltose barrier layer may be configured to allow glucose to pass through while substantially blocking maltose from passing through, such that the glucose oxidation current is above 10 nA / mMcm in steady state. 2 At the same level, the maltose oxidation current generated solely by maltose oxidation is less than 5 nA / mMcm. 2 .

[0042] A reference electrode can be configured to provide a potential reference level for the bias voltage applied to the glucose sensing electrode, regardless of whether the reduction of the chemical entity occurs in the reference electrode. In a three-electrode electrochemical cell, a counter electrode is provided in addition to the reference electrode for the reduction of the chemical entity therein, while in a two-electrode electrochemical cell, the reduction of the chemical entity occurs in the reference electrode.

[0043] In the aforementioned glucose sensing electrode, the nanoporous metal layer may include: an irregularly shaped substrate comprising a plurality of locally aggregated nanoparticles and interparticle gaps formed between adjacent nanoparticles within the irregularly shaped substrate, wherein the nanoparticles are generally elliptical or spherical with a diameter of about 2 nm to about 5 nm, and the interparticle gaps have an interparticle gap distance of about 0.5 nm to about 2 nm. Here, the irregularly shaped substrates may be interconnected to provide a three-dimensional interconnection network of irregularly shaped substrates. Irregularly shaped spaces may be formed between adjacent portions of the irregularly shaped substrates and are nanoscale or microscale in size, and the irregularly shaped spaces are interconnected to provide a three-dimensional interconnection network of irregularly shaped spaces.

[0044] Another aspect of the present invention provides a sensor device comprising: a single integrated body including a subcutaneous portion and a terminal portion; the subcutaneous portion including a glucose sensing electrode and a reference electrode, wherein when the subcutaneous portion is subcutaneously inserted into the body of a first subject, both electrodes are exposed to contact the tissue fluid of the first subject; and the terminal portion is configured to couple with a corresponding device and includes a first terminal electrically connected to the glucose sensing electrode and a second terminal electrically connected to the reference electrode. The glucose sensing electrode may include one or more features of the glucose sensing electrode described above.

[0045] Another aspect of the present invention provides a method for continuous glucose monitoring. The method includes: providing a sensor device; subcutaneously inserting a subcutaneous portion of a glucose sensing electrode into the body of a first subject, such that the glucose sensing electrode and a reference electrode are in contact with tissue fluid in the body of the first subject; applying a bias voltage of 0.2-0.45V relative to the reference electrode to the glucose sensing electrode; measuring the current generated by the glucose sensing electrode; calculating a glucose level using the current value obtained by measuring the current within less than one hour after subcutaneous insertion of the subcutaneous portion and application of the bias voltage; and displaying the calculated glucose level, within the range of about 4mM to about 20mM (approximately between about 72 mg / dL and about 360 mg / dL), as the glucose level of the first subject on a display. The glucose sensing electrode may include one or more of the features described above.

[0046] Another aspect of the present invention provides a sensor device comprising: a substrate; a first electrode (or a glucose sensing electrode) including a first conductive layer formed on the substrate and a glucose oxide layer formed on the first conductive layer; a first terminal formed on the substrate and electrically connected to the first electrode; a second electrode including a second conductive layer formed on the substrate; a second terminal formed on the substrate and electrically connected to the second electrode; a reference electrode including a third conductive layer formed on the substrate; and a third terminal formed on the substrate and electrically connected to the reference electrode.

[0047] In a sensor device, when a first electrode contacts a liquid containing glucose, ascorbic acid, and acetaminophen, and when a first bias voltage sufficient to oxidize glucose in a glucose oxide layer is applied between the first electrode and a reference electrode, the glucose oxide layer of the first electrode is configured to oxidize glucose and ascorbic acid therein with at least one of acetaminophen and is further configured to generate a first current, the first current comprising a glucose component generated by glucose oxidation and a first interfering component generated by the oxidation of at least one of ascorbic acid and acetaminophen in the glucose oxide layer. A second electrode is arranged in the device such that when the first electrode contacts the liquid, the second electrode also contacts the same liquid. The second electrode does not include a layer configured to oxidize glucose therein, such that when a second bias voltage is applied between the second electrode and the reference electrode, the second electrode is configured to oxidize ascorbic acid therein with at least one of acetaminophen but not glucose therein, and is further configured to generate a second current, the second current comprising a second interfering component generated by the oxidation of at least one of ascorbic acid and acetaminophen in the second electrode but not glucose. The device is configured to provide a first current at a first terminal and a second current at a second terminal.

[0048] The aforementioned sensor device can be configured to provide a second current connected to the first current when it provides a first current. The sensor device can be configured to simultaneously generate the first current and the second current. The sensor device can be configured to provide the first current and the second current, as well as information indicating the generation time of the first current and the second current. The sensor device can be configured to provide the second current and the first current when it provides the first current. In the aforementioned sensor device, the first current also includes a first background current generated by other electrochemical interactions between the liquid and the glucose sensing layer, wherein the second current also includes a second background current generated by other electrochemical interactions between the liquid and the second electrode.

[0049] In the aforementioned sensor device, when the first bias voltage is between 0.2V and 0.32V, the glucose oxide layer is configured to oxidize glucose and ascorbic acid without oxidizing acetaminophen, and the first interfering component is generated by the oxidation of ascorbic acid rather than the oxidation of acetaminophen. When the second bias voltage is between 0.2V and 0.32V, the second electrode is configured to oxidize ascorbic acid without oxidizing acetaminophen, and the second interfering component is generated by the oxidation of ascorbic acid rather than the oxidation of acetaminophen. In the aforementioned sensor device, when the first bias voltage is between 0.34V and 0.45V, the glucose oxide layer is configured to oxidize glucose, ascorbic acid, and acetaminophen, and the first interfering component is generated by the oxidation of ascorbic acid and acetaminophen. When the second bias voltage is between 0.34V and 0.45V, the second electrode is configured to oxidize ascorbic acid without oxidizing acetaminophen, and the second interfering component is generated by the oxidation of both ascorbic acid and acetaminophen.

[0050] In the aforementioned sensor device, the first electrode may further include a maltose barrier layer comprising polyphenylene diamine (poly-PD), the maltose barrier layer being formed on the glucose oxide layer. When in contact with a liquid containing glucose at a concentration of 4-20 mM (approximately 72-360 mg / dL) and when a bias voltage is applied, the maltose barrier layer is configured to allow glucose to pass through while substantially blocking maltose from passing through, such that, under steady-state conditions, the glucose oxidation current is above 10 nA / mMcm. 2 At the same level, the maltose oxidation current generated solely by maltose oxidation is less than 5 nA / mMcm. 2 .

[0051] The aforementioned sensor device may be a continuous glucose monitoring (CGM) electrode module, comprising a subcutaneous portion configured to subcutaneously contact a subject's bodily fluids, wherein a first electrode, a second electrode, and a reference electrode are formed in the subcutaneous portion. In the aforementioned sensor device, the glucose oxidation layer may include a nanoporous metal layer, wherein the first electrode may further include: an electrolyte ion blocking layer formed on the nanoporous metal layer and a biocompatible layer formed on the electrolyte ion blocking layer. The electrolyte ion blocking layer may be configured to inhibit Na+ contained in the liquid. + K + Ca 2+ Cl - PO4 3- and CO3 2- Diffusion into the nanoporous metal layer, so that Na exists between the top and bottom of the electrolyte ion barrier layer. + K + Ca 2+ Cl - PO43- and CO3 2- The general discontinuity of the combined concentration.

[0052] In the aforementioned sensor device, the electrolyte ion blocking layer may include a porous hydrophobic polymer layer configured to confine Na+ ions. + K + Ca 2+ Cl - PO4 3- and CO3 2- The electrolyte ion barrier layer may contain at least one of the following: poly(methyl methacrylate) (PMMA), poly(hydroxyethyl methacrylate) (PHEMA), and poly(methyl methacrylate-co-ethylene glycol dimethacrylate) (PMMA-EG-PMMA).

[0053] In the aforementioned sensor device, an electrolyte ion blocking layer may be configured to facilitate the regulation of the glucose sensing electrode, such that the regulation of the glucose sensing electrode is accomplished within 30 minutes after contact with the subject's body fluid by applying a bias voltage of 0.2-0.45V, wherein the regulation of the glucose sensing electrode is considered accomplished when either or both of the following conditions are met: when the rate of current decay is less than a first predetermined value and when the current remains less than a second predetermined value.

[0054] The aforementioned sensor device is a blood glucose monitoring (BGM) electrode module, comprising a reservoir configured to receive blood, wherein a first electrode, a second electrode, and a reference electrode are configured to contact the blood when blood is received in the reservoir. A first bias voltage is between 0.2V and 0.45V, wherein a second bias voltage is the same as or different from the first bias voltage. The glucose oxide layer may comprise a nanoporous metallic material or a glucose-specific enzyme configured to oxidize glucose. The glucose oxide layer may include an irregularly shaped body comprising a plurality of locally aggregated nanoparticles and interparticle gaps formed between adjacent nanoparticles within the irregularly shaped body, wherein the nanoparticles are generally elliptical or spherical with a diameter of about 2 nm to about 5 nm, and wherein the interparticle gaps have an interparticle gap distance of about 0.5 nm to about 2 nm. Here, the irregularly shaped bodies may be interconnected to provide a three-dimensional interconnection network of irregularly shaped bodies. Irregularly shaped spaces may be formed between adjacent portions of the irregularly shaped bodies and are nanoscale or microscale in size, and the irregularly shaped spaces are interconnected to provide a three-dimensional interconnection network of irregularly shaped spaces.

[0055] In another aspect, the present invention provides a system comprising: the aforementioned sensor device, further comprising a terminal portion in which first, second, and third terminals are arranged; a corresponding device comprising a first corresponding terminal, a second corresponding terminal, a third corresponding terminal, circuitry, and a power supply connected to the circuitry; and the corresponding device further comprising a corresponding terminal portion configured to connect to or engage with the terminal portion. Here, the first, second, and third corresponding terminals are arranged in the corresponding terminal portion such that when the terminal portion of the sensor device is connected to or engaged with the corresponding terminal portion of the corresponding device, the first terminal is electrically connected to the first corresponding terminal, the second terminal is electrically connected to the second corresponding terminal, and the third terminal is electrically connected to the third corresponding terminal. The circuitry of the corresponding device is configured to provide a first bias voltage between the first and third corresponding terminals, and the circuitry of the corresponding device is further configured to provide a second bias voltage between the second and third corresponding terminals.

[0056] In the above system, the corresponding device may include a wireless communication module configured to wirelessly communicate with a wirelessly paired computing device, the wirelessly paired computing device including at least one processor and at least one memory. The corresponding device may be configured to receive a first current at a first corresponding terminal and a second current at a second corresponding terminal. The corresponding device may be configured to transmit the second current and the first current while transmitting the first current, or the second current connected to the first current while transmitting the first current. The first current may be transmitted with a first timestamp, and the second current may be transmitted with a second timestamp, wherein the first and second timestamps indicate the same time.

[0057] The aforementioned system may also include software installed and executed by at least one processor of a wirelessly paired computing device. Upon execution, the software is configured to perform a method comprising the following steps: storing a first current and a second current received together or associated with each other from the corresponding device in the at least one memory of the computing device; processing the first current and the second current to provide a value indicating that glucose has been oxidized in the glucose oxide layer of the first electrode of the sensor device; and displaying the value or its corresponding information on a display of the computing device.

[0058] In the above system, either or both of the first current and the second current may be in the form of a continuous signal, wherein processing the first current and the second current may include processing the values ​​of the first current and the second current obtained simultaneously. Here, processing the value may include subtracting the second current from the first current. The first current and the second current may be stored in relation to each other in at least one memory. The above system may also include software installed and executed in a wirelessly paired computing device. After execution, the software is configured to perform data processing using the first current and the second current received from the corresponding device to obtain the glucose level contained in the liquid in contact with the first electrode of the sensor device. Here, the software requires the second current to obtain the glucose level during processing.

[0059] In the aforementioned system, the corresponding device may further include at least one processor, at least one memory, and software stored in the at least one memory and executable by the at least one processor. Upon execution, the software is configured to perform a method comprising the following steps: storing a first current and a second current received together or associated with each other from the sensor device in the at least one memory; and processing the first current and the second current to provide a value indicating that glucose has oxidized in the glucose oxide layer of the first electrode of the sensor device. Here, processing may include subtracting the second current from the first current. Either or both of the first current and the second current may be in the form of a continuous signal, wherein processing the first current and the second current may include processing the simultaneously obtained values ​​of the first current and the second current. The corresponding device may further include a display, wherein the method may further include displaying the value or its corresponding information on the display. The corresponding device may further include a wireless communication module configured to wirelessly pair with a device including the display, wherein the method may further include transmitting data to the wireless pairing device so that the value or its corresponding information is displayed on the display of the wireless pairing device.

[0060] In another aspect, the present invention provides an electrochemical sensing method. The method includes: providing a sensor device comprising a first electrode containing a glucose oxide layer capable of oxidizing glucose, a second electrode not containing a layer capable of oxidizing glucose, and a reference electrode; contacting the first, second, and reference electrodes in a liquid containing glucose, ascorbic acid, and acetaminophen; applying a first bias voltage sufficient to oxidize glucose in the glucose oxide layer between the first electrode and the reference electrode, such that at least one of glucose, ascorbic acid, and acetaminophen is oxidized in the glucose oxide layer and a first current is generated from the first electrode, wherein the first current includes a glucose component generated by the oxidation of glucose and a first interfering component generated by the oxidation of at least one of ascorbic acid and acetaminophen; applying a second bias voltage between the second electrode and the reference electrode, such that at least one of ascorbic acid and acetaminophen is oxidized in the second electrode but glucose is not oxidized therein and a second current is generated from the second electrode, wherein the second current includes a second interfering component generated by the oxidation of at least one of ascorbic acid and acetaminophen in the second electrode; and providing the first current and the second current for processing, wherein when the first current is provided for processing, the second current is also provided in connection with the first current.

[0061] In the above method, the first current and the second current can be generated simultaneously or sequentially within a reasonable period, wherein the glucose level remains substantially unchanged or exceeds a predetermined tolerance level. Information indicating the generation time of the first current and the second current, as well as information indicating the generation time of the second current, can be provided. The second current and the first current when the first current is provided can be provided. In the above method, a first bias voltage between 0.2V and 0.32V is applied to oxidize glucose and ascorbic acid in the glucose oxide layer without oxidizing acetaminophen, wherein the first interfering component is generated by the oxidation of ascorbic acid rather than the oxidation of acetaminophen; a second bias voltage between 0.2V and 0.32V is applied to oxidize ascorbic acid in the second electrode without oxidizing acetaminophen, wherein the second interfering component is generated by the oxidation of ascorbic acid rather than the oxidation of acetaminophen. In an alternative, a first bias voltage between 0.34V and 0.45V is applied to cause the glucose oxide layer to oxidize glucose, ascorbic acid, and acetaminophen, wherein the first interfering component is generated by the oxidation of ascorbic acid and acetaminophen; a second bias voltage between 0.34V and 0.45V is applied to cause the second electrode to oxidize ascorbic acid and acetaminophen, wherein the second interfering component is generated by the oxidation of both ascorbic acid and acetaminophen.

[0062] In the above method, the sensor device may further include a maltose barrier layer formed on the glucose oxide layer and comprising polyphenylene diamine (poly-PD). The sensor device may be a continuous glucose monitoring (CGM) electrode module comprising a subcutaneous portion configured to subcutaneously contact a subject's body fluids, wherein first, second, and reference electrodes are formed in the subcutaneous portion, wherein contacting the first, second, and reference electrodes with the liquid may include subcutaneously inserting the subcutaneous portion into the subject's body. The glucose oxide layer may include a nanoporous metal layer, wherein the first electrode may further include: an electrolyte ion barrier layer formed on the nanoporous metal layer and a biocompatible layer formed on the electrolyte ion barrier layer. The electrolyte ion barrier layer inhibits Na+ contained in the liquid. + K + Ca 2+ Cl - PO4 3- and CO3 2- Diffusion into the nanoporous metal layer, so that Na exists between the top and bottom of the electrolyte ion barrier layer. + K + Ca 2+ Cl - PO4 3- and CO3 2- The general discontinuity of the combined concentration.

[0063] In the above method, the sensor device is a blood glucose monitoring (BGM) electrode module including a reservoir, wherein contacting the first, second, and reference electrodes with the liquid may include providing a blood sample in the reservoir. The glucose oxide layer may include an irregularly shaped body comprising a plurality of locally aggregated nanoparticles and interparticle gaps formed between adjacent nanoparticles within the irregularly shaped body, wherein the nanoparticles are generally elliptical or spherical with a diameter of about 2 nm to about 5 nm, and wherein the interparticle gaps have an interparticle gap distance of about 0.5 nm to about 2 nm. The irregularly shaped bodies may be interconnected to provide a three-dimensional interconnection network of irregularly shaped bodies. Irregularly shaped spaces may be formed between adjacent portions of the irregularly shaped bodies and are nanoscale or microscale in size, and the irregularly shaped spaces may be interconnected to provide a three-dimensional interconnection network of irregularly shaped spaces.

[0064] In the above method, the sensor device may further include a first terminal electrically connected to a first electrode, a second terminal electrically connected to a second electrode, and a third terminal electrically connected to a reference electrode. The sensor device may also include a terminal portion in which the first, second, and third terminals are arranged, wherein applying a first bias voltage and a second bias voltage may include connecting a corresponding device, the corresponding device including a first corresponding terminal, a second corresponding terminal, a third corresponding terminal, circuitry, and a power supply connected to the circuitry. The corresponding device may further include a corresponding terminal portion for connecting or engaging the terminal portion of the sensor device. The first, second, and third corresponding terminals may be arranged in the corresponding terminal portion such that when the terminal portion of the sensor device is connected or engaged with the corresponding terminal portion of the corresponding device, the first terminal is electrically connected to the first corresponding terminal, the second terminal is electrically connected to the second corresponding terminal, and the third terminal is electrically connected to the third corresponding terminal. The circuitry of the corresponding device may provide a first bias voltage between the first and third corresponding terminals; the circuitry of the corresponding device may provide a second bias voltage between the second and third corresponding terminals.

[0065] In another aspect, the present invention provides a method for providing or measuring glucose levels. The method includes: providing software stored in at least one memory and executable by at least one processor, the memory and processor being provided in a sensor device or another device; executing the software with the at least one processor to process a first current and a second current to provide a value indicating oxidation of glucose in a glucose oxide layer of a first electrode of the sensor device; and displaying the value or corresponding information thereon on a display provided in the sensor device, other device, or another device.

[0066] In the above method, at least one memory and at least one processor are provided in other devices. The method may further include: transmitting a first current and a second current to other devices; and, prior to execution, storing the first current and the second current received together or in association with each other in at least one memory. In the above method, the first current is transmitted with a first timestamp, and the second current is transmitted with a second timestamp, wherein the first and second timestamps indicate the same time. In the above method, either or both of the first current and the second current may be in the form of a continuous signal, wherein processing the first current and the second current may include processing the values ​​of the first current and the second current obtained simultaneously. In the above method, processing may include subtracting the second current from the first current.

[0067] Another aspect of the present invention provides a sensor device comprising: a working electrode including a nanoporous metal layer; a reference electrode; and a bias voltage applied between the working electrode and the reference electrode, wherein a glucose-specific enzyme is absent in the working electrode.

[0068] In a sensor device, a nanoporous metal layer includes an irregularly shaped body comprising a plurality of locally aggregated nanoparticles and interparticle gaps formed between adjacent nanoparticles within the irregularly shaped body. The nanoparticles are generally elliptical or spherical, having a diameter of about 2 nm to about 5 nm, and the interparticle gaps have an interparticle distance of about 0.5 nm to about 2 nm. The irregularly shaped bodies can be interconnected to provide a three-dimensional interconnection network. Irregularly shaped spaces are formed between adjacent portions of the irregularly shaped bodies and are nanoscale or microscale in size, and these irregularly shaped spaces are interconnected to provide a three-dimensional interconnection network. In the sensor device, a bias voltage is set sufficient to oxidize glucose in the nanoporous metal layer but insufficient to oxidize acetaminophen in the nanoporous metal layer, wherein the bias voltage is set in the range of about 0.20 V to about 0.32 V.

[0069] The sensor device may include a continuous glucose monitoring (CGM) electrode module comprising a subcutaneous portion configured for subcutaneous contact with a subject's bodily fluids, wherein a working electrode and a reference electrode are formed in the subcutaneous portion. The working electrode may further include: an electrolyte ion blocking layer formed on a nanoporous metal layer; and a biocompatible layer formed on the electrolyte ion blocking layer. The electrolyte ion blocking layer may be configured to inhibit sodium content in the liquid. + K + Ca 2+ Cl - PO4 3- and CO3 2- Diffusion into the nanoporous metal layer, so that Na exists between the top and bottom of the electrolyte ion barrier layer. + K + Ca 2+ Cl - PO4 3- and CO3 2- The combined concentrations exhibit a general discontinuity. An electrolyte ion-blocking layer can be configured to facilitate the modulation of the working electrode, such that modulation is achieved within 30 minutes of contact with the subject's bodily fluids by applying a bias voltage.

[0070] The aforementioned sensor device may further include a maltose barrier layer comprising polyphenylene diamine (poly-PD) and interposed between a nanoporous metal layer and an electrolyte ion barrier layer. When in contact with a liquid containing maltose and glucose at a concentration of 4-20 mM (approximately 72-360 mg / dL) and when a bias voltage is applied, the maltose barrier layer is configured to allow glucose to pass through while substantially preventing maltose from passing through, such that, under steady-state conditions, the glucose oxidation current is above 10 nA / mMcm. 2At the same level, the maltose oxidation current generated solely by maltose oxidation is less than 5 nA / mMcm. 2 .

[0071] In another aspect, the present invention provides a glucose sensing method. The method includes: providing one of the aforementioned sensor devices; and applying a bias voltage between a working electrode (or glucose sensing electrode) and a reference electrode in the range of about 0.20V to about 0.32V. Here, the application of the bias voltage causes glucose to oxidize in a nanoporous metal layer, such that the glucose oxidation current generated solely by glucose oxidation is above 10 nA / mMcm. 2 At a certain level, the applied bias voltage prevents sufficient oxidation of acetaminophen in the nanoporous metal layer, ensuring that the acetaminophen oxidation current generated by the oxidation of acetaminophen in the nanoporous metal layer is below 5 nA / mMcm. 2 . Attached Figure Description

[0072] The patent or application documents include color drawings. Upon request and payment of the necessary fees, a published copy of the patent or patent application with color drawings will be provided by the Patent Office.

[0073] Figure 1 A conceptual electrochemical glucose sensing system according to an embodiment of the present invention is shown.

[0074] Figure 2 The working electrode of an enzyme glucose sensing system according to one embodiment is shown.

[0075] Figure 3 A working electrode comprising a nanoporous layer is shown in a non-enzymatic sensing system according to one embodiment.

[0076] Figure 4 The top surface and depth of the nanoporous layer are shown.

[0077] Figure 5A The cluster-like morphology of a nanoporous layer according to one embodiment is shown.

[0078] Figure 5B These are TEM images of a cluster based on an implementation scheme.

[0079] Figure 5C yes Figure 5B A magnified image of a TEM photograph.

[0080] Figure 5D It is a SEM image of a nanoporous layer taken from its top according to one embodiment.

[0081] Figure 6AThis is a flowchart for manufacturing a clustered nanoporous layer according to one embodiment.

[0082] Figure 6B This is a flowchart for manufacturing a clustered nanoporous layer according to another embodiment.

[0083] Figure 7 This is an exemplary phase diagram showing different phases of surfactants.

[0084] Figure 8 An antimicelle phase and a nanoparticle-surfactant colloid are shown according to one embodiment.

[0085] Figure 9 Including TEM images of nanoparticle clusters according to one embodiment.

[0086] Figure 10A The non-clustered morphology of a nanoporous layer according to one embodiment is shown.

[0087] Figure 10B It is a TEM image of the non-clustered morphology of a nanoporous layer formed on a metal surface according to an embodiment.

[0088] Figure 11 This is a flowchart for manufacturing a non-clustered nanoporous layer according to one embodiment.

[0089] Figure 12 This is a flowchart for manufacturing a hexagonal nanostructure according to one implementation scheme.

[0090] Figure 13A The formation of a hexagonal arrangement according to one embodiment is shown.

[0091] Figure 13B Metal deposition using a hexagonal arrangement of liquid crystal phases is shown.

[0092] Figure 14 The particle size distribution of a nanoparticle-surfactant colloid prepared according to one embodiment is shown.

[0093] Figure 15 The particle size distribution of a clustered colloid prepared according to one embodiment is shown.

[0094] Figure 16A and 16B Cross sections of the electrode substrate and the non-enzymatic glucose sensing working electrode according to the embodiment are shown respectively.

[0095] Figures 17A-17C This is an SEM image of the glucose sensing working electrode according to the implementation scheme.

[0096] Figure 18This is a distribution diagram of the current generated by the oxidation of glucose and other materials in PBS according to the implementation plan.

[0097] Figure 19 It is a distribution diagram of the current generated by the oxidation of glucose and other materials in human serum according to the implementation plan.

[0098] Figure 20 It is the structural formula of the maltose molecule.

[0099] Figure 21 A non-enzymatic working electrode including a maltose barrier layer is shown according to one embodiment.

[0100] Figure 22 A scan of the oxidation voltage during the cyclic voltammetric electrochemical polymerization of phenylenediamine is shown according to the embodiment.

[0101] Figure 23 A chronoamperometry setup for electroshock treatment to adjust the porosity of a porous polymer layer is shown according to one embodiment.

[0102] Figure 24 This is a flowchart for manufacturing a maltose barrier layer according to one embodiment.

[0103] Figure 25-30 The current monitored using a glucose sensing electrode with a maltose barrier layer according to the embodiment is shown, wherein the current signals are represented in color because they are not easily seen in black and white film.

[0104] Figure 31 A CGM working electrode according to one embodiment is shown.

[0105] Figure 32 The decrease in electrolyte concentration is shown with respect to the thickness of the electrolyte ion barrier layer according to one embodiment.

[0106] Figure 33 A CGM electrode unit according to one embodiment is shown.

[0107] Figure 34 This is a flowchart for manufacturing a CGM electrode unit according to one implementation scheme.

[0108] Figures 35-37 It shows the manufacturing process. Figure 33 Top view and cross-sectional view of intermediate products at various stages of the CGM electrode, wherein each cross-section is cut along line 3501 and viewed in the direction of the arrow.

[0109] Figure 38A and 38BCross-sections of intermediate products after the formation of nanoporous layers and CGM working electrodes with functional layers, according to the embodiments, are shown.

[0110] Figure 39 A disposable glucose sensing kit according to an embodiment is shown.

[0111] Figure 40 A two-electrode glucose sensing system according to one embodiment is shown.

[0112] Figure 41 A CGM electrode unit of a two-electrode glucose sensing system according to one embodiment is shown.

[0113] Figure 42A It is a current distribution diagram generated by glucose oxidation according to one embodiment, wherein the working electrode does not include an electrolyte ion blocking layer. Figure 42B yes Figure 42A A magnified view of a portion of the distribution map.

[0114] Figure 43 It is a current distribution diagram generated by glucose oxidation according to one embodiment, wherein the working electrode includes an electrolyte ion blocking layer.

[0115] Figure 44 It is a comparison of the adjustment time of the working electrode with and without an electrolyte ion barrier layer.

[0116] Figure 45A , 45B The image shows a voltage regulator according to one implementation scheme, and 45C is a photograph of it.

[0117] Figure 46 This is a diagram illustrating CGM monitoring of glucose levels in rats using a non-enzymatic CGM electrode module according to one embodiment.

[0118] Figure 47 It is a Clarke Error Grid for a non-enzymatic CGM electrode module according to one implementation scheme.

[0119] Detailed description of the implementation plan

[0120] The subject matter of this disclosure will now be described and discussed in more detail with reference to the accompanying drawings, which illustrate some, but not all, embodiments of the invention. Similar numerals throughout denote similar elements or portions. The subject matter of this disclosure may be embodied in many different forms and should not be construed as limited to the specific embodiments listed herein. Rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the subject matter of this disclosure will be apparent to those skilled in the art. Therefore, it should be understood that the subject matter of this disclosure is not limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.

[0121] Electrochemical glucose sensing system

[0122] Electrochemical glucose detection

[0123] Electrochemical glucose sensing measures the glucose concentration in an electrolyte solution. Figure 1 An electrochemical glucose sensing system 101 for detecting glucose concentration in a test fluid or electrolyte solution 102 is conceptually illustrated. System 101 includes a working or sensing electrode 103, a counter electrode 105, and a reference electrode 106, which are connected to a voltage regulator 104 and in contact with the test fluid 102. In an embodiment, the voltage regulator includes circuitry serving as a voltage source 109 and a current sensor 108. The voltage source 109 provides a bias voltage that drives redox reactions at the working electrode 103 and the counter electrode 105. The voltage regulator also includes circuitry, such as an operational amplifier 107, for maintaining the bias voltage on the working electrode 103 relative to the reference electrode 106. The current sensor 108 detects the current generated by the redox reactions involving glucose contained in the test fluid 102.

[0124] Enzyme glucose sensing electrode

[0125] Most (but not all) electrochemical glucose sensing systems use glucose-specific enzymes to detect glucose molecules. Figure 2 A working electrode 103E of an enzyme glucose sensing system (i.e., an enzyme glucose sensing electrode) is shown. The terms "glucose sensing electrode" and "working electrode" are used interchangeably in this disclosure. The enzyme working electrode 103E includes a conductive layer 110 and an enzyme layer 111. Optionally, the enzyme working electrode 103E may include at least one functional layer 112 on the enzyme layer 111, such as... Figure 2As shown. Alternatively, although not shown, at least one functional layer may be located between the enzyme layer 111 and the conductive layer 110. The enzyme layer 111 contains glucose-specific enzyme molecules 115, which are held therein by a fixator 113. When glucose molecules come into contact with the glucose-specific enzyme, the enzyme catalyzes the oxidation of glucose to gluconolactone. Electrons from the oxidation of glucose are ultimately transferred to the conductive layer 110 to generate a current in the circuitry of the electrochemical sensing system 101.

[0126] glucose oxidase

[0127] In some enzyme glucose sensing systems, the enzyme working electrode 103E includes glucose oxidase (GOx). Glucose oxidase 115 transfers electrons to molecular oxygen residing near the enzyme, and the molecular oxygen is reduced to hydrogen peroxide. Under an appropriate bias voltage applied to the system, the conductive layer 110 oxidizes the hydrogen peroxide and gains electrons from it, thereby generating a current that indicates the glucose concentration in the test fluid 102.

[0128] glucose dehydrogenase

[0129] In other enzyme glucose sensing systems, the enzyme working electrode 103E includes glucose dehydrogenase (GDH). Unlike glucose oxidase, glucose dehydrogenase does not use oxygen but instead transfers electrons to other adjacent chemical entities called electron mediators, which then transfer electrons from glucose oxidation to conductive layer 110. The electron mediators may be contained within enzyme layer 111. Alternatively, the electron mediators may be provided in an insulating layer (not shown) between enzyme layer 111 and conductive layer 110. While glucose dehydrogenase has some advantages over glucose oxidase, this enzyme oxidizes both maltose and glucose, which interferes with accurate sensing of glucose concentration.

[0130] Non-enzymatic glucose sensing electrode

[0131] Non-enzymatic electrochemical glucose sensing systems do not use glucose-specific enzymes or any enzymes used to detect glucose. Instead, they have a non-enzymatic working electrode that detects glucose in the absence of a glucose-specific enzyme. In an embodiment, the non-enzymatic working electrode includes at least one glucose oxide layer capable of oxidizing glucose molecules at a moderate bias voltage. Generally, the higher the bias voltage, the more likely glucose oxidation will occur in at least one glucose oxide layer. However, there are limitations on the bias voltage because other chemical entities will also be oxidized at high bias voltages. Therefore, non-enzymatic electrochemical glucose sensing relies on a material that oxidizes glucose at a bias voltage that does not oxidize other chemical entities contained in the test fluid.

[0132] Nanoporous layers for non-enzymatic glucose sensing electrodes

[0133] Figure 3 A non-enzymatic working electrode (hereinafter referred to as the "working electrode") 103NE is shown, comprising a conductive layer 110 and a nanoporous glucose oxidation layer (or nanoporous layer) 117. In an embodiment, the nanoporous layer 117 includes a nanoporous internal structure that enables, facilitates, or promotes the oxidation of glucose at a moderate bias voltage. When glucose is oxidized, the conductive layer 110 gains electrons from the glucose oxidation and generates a current in the circuit. The current can be detected by a current sensor 108 and interpreted by the system's hardware and software. Optionally, the working electrode 103NE may include at least one functional layer 112 on or between the nanoporous layer 117 and the conductive layer 110 (not shown).

[0134] Conductive layer - material

[0135] Under bias voltage Figure 2 and Figure 3 The conductive layer 110 in the system acquires electrons from the glucose oxidation reaction and transfers them to the current sensor 108. In embodiments, the conductive layer 110 comprises or is made of at least one conductive material and is connected to the circuitry of the system 101. In some embodiments, given the small scale of the conductive layer 110, a semiconducting material may be used instead of the conductive material. Non-limiting examples of conductive layer materials include platinum (Pt), gold (Au), silver (Ag), ruthenium (Ru), stainless steel, silicon (amorphous, polycrystalline, and single-crystal), and conductive carbon materials, including graphite, graphene, fluorene, and carbon nanotubes. In the embodiments described, the conductive layer 110 does not include the nanoporous internal structure of the glucose oxide layer 117.

[0136] Conductive layer - configuration

[0137] In some embodiments, the conductive layer 110 may be formed from a single layer of homogeneous material. Alternatively, the conductive layer 110 may comprise sublayers made of multiple different materials. In some embodiments, the conductive layer 110 includes a top sublayer and one or more sublayers beneath it. In some embodiments, the top sublayer does not contain silver, copper, aluminum, or other conductive materials that are more easily oxidized than silver, copper, or aluminum. The top sublayer may be less conductive than the other sublayers. In some embodiments, the conductive layer 110 includes a conductive carbon layer as a top sublayer and a silver layer as another sublayer beneath it. The conductive layer 110 has a thickness that can vary significantly depending on the specific instance. In some embodiments, the conductive layer 110 may be omitted, and the nanoporous layer is directly connected to the current sensor via conductive wires or connecting wires.

[0138] Reverse electrode

[0139] Under a bias voltage, reduction of the chemical entity occurs at the counter electrode 105. In an embodiment, the counter electrode 105 comprises at least one conductive or semiconductive material and is connected to the circuitry of system 101. In an embodiment, the counter electrode 105 may be formed of a single layer of homogeneous material or multiple layers of different materials. The conductive or semiconductive material of the conductive layer 110 may also be used in the counter electrode 105, although in a particular system, the conductive layer 110 and the counter electrode 105 may use different materials.

[0140] Reference electrode

[0141] The reference electrode 106 provides stability in the electrochemical sensing system by maintaining a bias voltage between the sensing electrode 103 and the reference electrode. Therefore, glucose oxidation can continue at the sensing electrode 103 even if the reduction rate at the counter electrode 105 differs from the oxidation rate at the sensing electrode 103. In some embodiments, the counter electrode 105 may be omitted, and the reference electrode 106 may function as both a counter and a reference electrode. In some embodiments, the reference electrode 106 may be formed from a single layer of homogeneous material or multiple layers of different materials. The conductive or semi-conductive material of the conductive layer 110 may also be used in the reference electrode 105, although in a particular system, the conductive layer 110 and the reference electrode 106 may use different materials. In some embodiments, the reference electrode 106 may include a salt layer on a layer of conductive or semi-conductive material. For example, the salt layer may be made of or include silver chloride (AgCl).

[0142] Current sensor

[0143] Current sensor 108 measures the current flowing from working electrode 103. Current sensor 108 can detect the current flowing at a specific point in time using the amperometric method. Alternatively, current sensor 108 can be a quantity measuring device.

[0144] Test fluid

[0145] In some embodiments, the test fluid is a biological fluid of a human or animal, but is not limited thereto. In some embodiments, the test fluid is a liquid mixture comprising a biological fluid and at least one additional substance added to the biological fluid. Biological fluids include, but are not limited to, blood, tissue fluid, cerebrospinal fluid, lymph, or urine. In some embodiments, the test fluid includes a non-biological fluid prepared for an experiment.

[0146] bias voltage

[0147] The bias voltage applied between the working electrode 103NE and the reference electrode 106 is or is approximately 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, or 0.46 V. In the implementation, the applied bias voltage can be within a range formed by selecting any two numbers (two voltage values) listed in the preceding sentence, for example, between about 0.20V and about 0.30V, between about 0.30V and about 0.40V, between about 0.28V and about 0.40V, between about 0.30V and about 0.38V, between about 0.28V and about 0.36V, and so on.

[0148] Nanoporous layers

[0149] Nanoporous layers

[0150] The nanoporous layer 117 of the working electrode 103NE includes nanoscale internal structures such as cavities, spaces, and openings (collectively referred to as "nanopores"). In embodiments, the nanopores of the nanoporous layer 117 enable or facilitate glucose oxidation, and glucose concentration can be measured based on the current generated by glucose oxidation. While no aspect of the invention is bound by any theory or concept, it is conceivable that glucose oxidation occurs when glucose molecules enter the nanopores and come into contact with the inner surface of the nanoporous layer 117 more frequently and for longer than with the non-porous surface of the electrode.

[0151] Enzyme-free and electron mediator-free

[0152] By incorporating the nanoporous layer 117, a glucose-specific enzyme-free working electrode 103NE can be provided, which requires a more complex manufacturing process and has lower stability than the solid-state material of the nanoporous layer 117. Furthermore, the enzyme-sensing electrode 103NE can operate without electron mediators that facilitate electron transfer between different materials. In an embodiment, the working electrode 103NE contains neither an enzyme nor an electron mediator.

[0153] Materials with nanoporous layers

[0154] In some embodiments, the nanoporous layer 117 is made of, or includes but is not limited to, platinum (Pt), gold (Au), palladium (Pd), rhodium (Rh), titanium (Ti), ruthenium (Ru), tin (Sn), nickel (Ni), copper (Cu), indium (In), thallium (Tl), zirconium (Zr), iridium (Ir), or oxides of the foregoing. In other embodiments, the nanoporous layer 117 is made of, or includes, an alloy of two or more metallic elements listed in the preceding sentence, including but not limited to Pt-Ir, Pt-Ru, and Pt-Pd.

[0155] Roughness coefficient definition

[0156] Roughness coefficient, or roughness, is the ratio of an object's actual surface area to its geometric surface area. Here, geometric surface area refers to the object's projected area onto a plane, disregarding its internal surfaces. Actual surface area refers to the total surface area, taking into account internal surfaces. See also... Figure 4 For example, if the nanoporous layer 117 is a rectangular block with a height or depth 118 and a top rectangle 119, the projected area or geometric surface area of ​​the nanoporous layer is the area of ​​the top rectangle exposed to the outside. The actual surface area of ​​the nanoporous layer can be measured, for example, using a known cyclic voltammetry technique that detects the current from proton adsorption on the actual surface using an electrochemical method.

[0157] Roughness coefficient of nanoporous layers

[0158] The roughness coefficient value represents the total amount of internal pores within the nanoporous layer 117. The roughness coefficient of the nanoporous layer 117 can be related to its sensitivity to glucose oxidation. Generally, a higher roughness coefficient indicates that more glucose oxidation may occur. The roughness coefficient of the nanoporous layer 117 is, or approximately, 100, 200, 300, 400, 500, 600, 700, 800, 900, 100, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, or 2500. In the implementation, the roughness coefficient can be within a range formed by selecting any two numbers (two roughness coefficient values) listed in the preceding sentence, for example, between about 100 and about 2500, between about 750 and about 1250, or between about 850 and about 1150.

[0159] Thickness of nanoporous layer

[0160] The roughness coefficient value does not represent the porosity or density level of the nanoporous material per unit volume, but rather the total amount of internal pores. Therefore, depending on the porosity level of the nanoporous material, the thickness of the nanoporous layer can be adjusted in embodiments to achieve a target roughness coefficient value. In embodiments, the thickness of the nanoporous layer 117 can be about 0.03, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 μm. In some embodiments, the thickness can be within a range formed by selecting any two numbers (two thickness values) listed in the preceding sentence, for example, between about 0.05 μm (50 nm) and about 10 μm, between about 0.5 μm and about 8 μm, or between about 2 μm and about 7 μm.

[0161] form

[0162] The nanoporous layer 117 may have different internal morphologies in each specific fabrication. In some embodiments, the nanoporous layer 117 may include or be made of nanoparticles deposited together, forming nanopores (intergranular nanopores) within themselves. In other embodiments, the nanoporous layer 117 may include or be made of clusters of nanoparticles deposited together, the nanoparticles forming intergranular nanopores within the clusters and also forming spaces (interclusive gaps or spaces) within the clusters. In other embodiments, the nanoporous layer 117 may include or be made of repeating a specific shape, including nanostructures of nanopores such as hexagonal structures. Moreover, in each specific fabrication, the nanoporous layer 117 may have different porosity levels and different roughness coefficient values ​​per unit volume.

[0163] Fabrication of nanoporous layers

[0164] The nanoporous layer 117 can be prepared using a liquid composition containing metal ions and a surfactant. In embodiments, different morphologies of the nanoporous layer can be formed using different phases of the surfactant. A micellar phase, an anti-micllar phase, a liquid crystal phase, or another phase of the surfactant can be used to produce a nanoporous layer with a specific morphology. In these different phases, the metal ions are arranged in a row or locally concentrated next to the hydrophilic portion of the surfactant. The metal ions positioned in the liquid composition undergo additional processes of reduction and deposition on the surface to provide nanoporous layers 117 with different morphologies.

[0165] Clustered nanoporous layers

[0166] Clustered morphology

[0167] Figure 5AThis is a diagram of a vertical cross-section of a nanoporous layer 120 with a clustered morphology on a substrate 129. In reality at the nanoscale, the top surface of the substrate 129 may not be as flat as shown and may be uneven. In the clustered morphology 120, many nanoparticles 121 aggregate together to form irregularly shaped clusters 125. For ease of illustration, different shading or shading lines are used for the different clusters 125. These irregularly shaped clusters 125 are irregularly stacked together to form the nanoporous layer. Figure 5B These are transmission electron microscopy (TEM) images of some clusters 125 before they are deposited to form a nanoporous layer. Figure 5C yes Figure 5B A magnified image of the ring-shaped portion. Figure 5D It is a scanning electron microscope (SEM) image of a nanoporous layer with a clustered morphology, taken from the top of the nanoporous layer.

[0168] Cluster-like pores and spaces

[0169] In the irregular stacking of irregularly shaped clusters 125, adjacent clusters form inter-cluster gaps or spaces 127 between them. These inter-cluster gaps 127 can be nanoscale or microscale. In this disclosure, nanoscale means greater than 1 nm and less than 100 nm, while microscale means greater than 100 nm and less than 100 μm. Each cluster 125 includes or is made of generally spherical or elliptical nanoparticles 121. Within each cluster, individual nanoparticles are typically separated from each other and form small gaps 123 therebetween. These small gaps are nanoscale and are referred to as interparticle nanopores 123. In embodiments, interparticle nanopores are distributed throughout the clusters. In embodiments, interparticle nanopores form interconnected or networked channels within each cluster. Figure 5A and 5D These interparticle nanopores 123 are shown in each cluster 125.

[0170] Formation of inter-cluster gaps / spaces

[0171] In this embodiment, to produce a clustered morphology, irregularly shaped clusters 125 are first prepared as a suspension in a liquid. The suspension is then distributed onto a substrate 129, which is then dried. As the liquid dries, the clusters spontaneously deposit onto the substrate and other clusters. No external force is applied to the clusters during drying. Therefore, the clusters are not compacted during deposition. As the clusters deposit and stack, each cluster may contact the substrate surface or adjacent clusters. After drying, the clusters are adjacent to or in contact with adjacent or neighboring clusters. The deposited clusters are interconnected or integrated together via adjacency and contact. Due to the irregular shape of the individual clusters, irregular gaps and spaces are formed between adjacent clusters, wherein the gaps and spaces define the irregular shape of the deposited clusters as if the surface and contour of the deposited clusters were surrounded by irregular gaps and spaces. These irregular gaps and spaces are referred to as inter-cluster gaps or spaces 127.

[0172] Distribution of clusters and inter-cluster gaps

[0173] In this embodiment, irregularly shaped clusters 125 are distributed throughout the clustered morphology 120 of the nanoporous layer 117. The irregularly shaped clusters 125 are interconnected via adjacency, meaning that these clusters contact themselves and form a three-dimensional network of clusters generally throughout the nanoporous layer 117. Inter-cluster gaps 127 define and surround the surfaces of the irregularly shaped clusters and are interconnected to form three-dimensional interconnected or networked channels throughout the nanoporous layer 117. The inter-cluster gaps and spaces 127 are well distributed throughout the nanoporous layer 117 from top (not shown) to bottom (on or adjacent to substrate 129). The three-dimensional network of irregularly shaped clusters and the three-dimensional network of irregularly shaped gaps are three-dimensionally complementary to form a highly networked three-dimensional mesh structure. The three-dimensional network of clusters and channels can resemble the three-dimensional internal shape of a sponge, except that the inter-particle gaps and spaces are networked together throughout the nanoporous layer 117.

[0174] Distribution of nanoparticles and interparticle nanopores

[0175] Given that each cluster is formed of numerous nanoparticles 121 and interparticle nanopores 123, the nanoparticles 121 and interparticle nanopores 123 are typically distributed throughout the nanoporous layer 117. Therefore, the interparticle nanopores 123 are interconnected within each cluster and with the interparticle nanopores of other clusters, which are typically distributed throughout the nanoporous layer 117 via the interparticle nanopores in adjacent clusters and via the inter-cluster gaps 127 interconnected throughout the nanoporous layer 117.

[0176] Intercluster gaps / spaces for glucose diffusion

[0177] In this embodiment, the interconnection of the inter-cluster gaps 127 provides a networked channel for the diffusion of glucose molecules (0.7-0.8 nm long) within the nanoporous layer 117. It should be understood that glucose oxidation primarily occurs in the nanopores between nanoparticles, rather than in spaces at the micrometer scale. Because the inter-cluster gaps 127 are networked or interconnected throughout the nanoporous layer 117, glucose molecules can reach virtually anywhere within the nanoporous layer 117 via the inter-cluster spaces, which are large-scale considering the size of the glucose molecule. Furthermore, due to the good interconnection between the inter-cluster gaps 127 and the inter-particle nanopores 123, the inter-particle nanopores 123 at any location within the nanoporous layer 117 can be exposed and opened for glucose oxidation. Therefore, compared to a nanoporous layer without such interconnected channels formed by the inter-cluster gaps, the three-dimensional interconnection or networked channels of the inter-cluster gaps can provide more glucose oxidation, i.e., a stronger signal (higher current) for glucose oxidation.

[0178] Two types of particles and two types of pores

[0179] As discussed, the cluster morphology 120 comprises two different types of particles that define two different types of pores. In terms of particles, one type is nanoparticles 121, and the other is clusters 125 composed of nanoparticles 121. In terms of pores, one type is interparticle nanopores 123 between nanoparticles 121 within clusters 125, and the other is interclusard spaces 127 between clusters 125.

[0180] Clusters of nanoparticles

[0181] Figure 5BTEM images show irregularly shaped clusters. The number of nanoparticles 121 in each cluster can vary widely, and the size of the clusters 125 can vary accordingly. In the cluster morphology, some clusters 125 are nanometer-sized (less than 100 nm), while others are micrometer-sized (100 nm to 100 μm). Clusters 125 have the following lengths or diameters: approximately 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, or 700 nm. In an embodiment, the length or diameter of cluster 125 may be within a range formed by selecting any two numbers (two length or diameter values) listed in the preceding sentence, for example, between about 20 nm and about 300 nm, or between about 60 nm and about 240 nm. Cluster 125 may have the following average diameter or length: about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 280, or 300 nm. In an embodiment, the average diameter of cluster 125 may be within a range formed by selecting any two numbers listed in the preceding sentence, for example, between about 100 nm and about 220 nm.

[0182] Nanoparticles

[0183] Figure 5C The TEM images show nanoparticles within a single cluster. The nanoparticles 121 within the cluster are discrete and generally spherical (globose) or elliptical (oval), but are not limited thereto. The nanoparticles 121 have diameters of approximately 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, or 6.5. In embodiments, the diameter can be within a range formed by selecting any two numbers (two diameter values) listed in the preceding sentence, for example, between approximately 2 nm and approximately 5 nm. The nanoparticles 121 may have an average diameter of approximately 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, or 4.0. In one embodiment, the average diameter of the nanoparticles 121 may be within a range formed by selecting any two numbers listed in the preceding sentence, for example, between about 2.5 nm and about 4.0 nm, between about 2.75 nm and about 3.75 nm, or between about 2.25 nm and about 3.5 nm. In another embodiment, nanoparticles with an average diameter of 2-5 nm are distributed throughout the nanoporous layer 117.

[0184] interparticle nanopores

[0185] Figure 5C The TEM images also show interparticle nanopores between nanoparticles within the cluster. These interparticle nanopores are networked and interconnected within the cluster. The interparticle gaps or nanopores 123 have interparticle gap distances between two adjacent nanoparticles within the same cluster. The interparticle gap distances are approximately 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.5, 3.0, 3.5, 4.0, or 4.5 nm. In embodiments, the interparticle gap distance can be within a range formed by selecting any two numbers (two distance values) listed in the preceding sentence, for example, between approximately 0.5 nm and approximately 4.5 nm, or between approximately 1.5 nm and approximately 4.0 nm. The interparticle nanopores 123 may have the following average interparticle spacing distances: about 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, or 3.5 nm. In an embodiment, the average interparticle spacing distance of the nanopores 123 may be within a range formed by selecting any two numbers listed in the preceding sentence, for example, between about 0.75 nm and about 1.5 nm, or between about 1.0 nm and about 2.5 nm. In an embodiment, the interparticle nanopores 123 having an average interparticle spacing distance of 1-2.5 nm are distributed throughout the nanoporous layer 117.

[0186] Interclusory space

[0187] Figure 5D The SEM images show the openings between the networked clusters visible from the top of the nanoporous layer. Although the three-dimensional shape is not well represented... Figure 5DIn the two-dimensional image, the top surface of the nanoporous layer includes valleys and hills formed by stacked clusters. Within the nanoporous layer, valleys and hills form inter-cluster gaps. These inter-cluster gaps, or spaces, are irregularly shaped. The inter-cluster gaps 127 are in the nanometer to micrometer range. The inter-cluster gaps 127 have the following inter-cluster gap distances: approximately 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, or 700 nm. In an embodiment, the inter-cluster gap distance can be within a range formed by selecting any two numbers listed in the preceding sentence, for example, between approximately 100 nm and approximately 1000 nm. The inter-cluster gap 127 has the following average inter-cluster gap distance: approximately 100, 150, 200, 250, 300, 350, 400, 450, or 500 nm. In an embodiment, the average inter-cluster gap distance can be within a range formed by selecting any two numbers listed in the preceding sentence, for example, between approximately 150 nm and approximately 400 nm.

[0188] Fabrication of clustered nanoporous layers

[0189] The whole process

[0190] In this implementation, an isotropic reverse micelle phase (or "reverse micelle phase") of a surfactant can be used to prepare a nanoporous layer with a clustered morphology. See also Figure 6A In step 601, an aqueous liquid composition is prepared in a reverse micelle phase using a metal ion source and a surfactant. The metal ions are locally concentrated within the hydrophilic spaces of individual reverse micelles. Subsequently, in step 603, a reducing agent is added to the reverse micelle phase to form metal nanoparticles (“nanoparticle colloids” or “nanoparticle-surfactant colloids”) dispersed in the surfactant-containing liquid composition. Subsequently, in step 605, the surfactant is removed from the nanoparticle-surfactant colloids, and clusters of nanoparticles dispersed in the liquid are collected (“cluster colloids” or “cluster-liquid colloids”). Optionally, in step 607, the collected cluster colloids are mixed with a non-surfactant liquid. In step 609, the cluster colloids are dispensed onto a surface, for example by printing without electroplating. Subsequently, in step 611, the liquid is dried, thereby forming a nanoporous layer 117 on surface 129.

[0191] surfactants

[0192] Surfactants are amphiphilic organic compounds that have a hydrophilic head (or hydrophilic moiety) and a hydrophobic tail (hydrophobic moiety) in a single molecule. Surfactants can form different structures or phases in water depending on their concentration and temperature. Figure 7 An exemplary phase diagram is shown, comprising a surfactant comprising the following different phases: micelle phase 131, hexagonal phase 133, lamellar phase 135, and two micelle phases 137.

[0193] Preparation of isotropic antimicelle phase

[0194] In step 601, an isotropic reverse micelle phase is prepared using an aqueous liquid composition containing a surfactant, metal ions, and water. For example... Figure 8 As shown in the conceptual diagram, the reverse micelle phase comprises reverse micelles 141 formed from surfactant molecules. Each reverse micelle 141 includes a hydrophilic core 143 surrounded by a hydrophobic tail radiating from the hydrophilic core. The hydrophilic core 143 comprises the hydrophilic components of the liquid composition, namely water and metal ions. Therefore, the metal ions are locally concentrated within the hydrophilic core 143 of the reverse micelle.

[0195] Examples of surfactants

[0196] The surfactant is selected from those surfactants that can form an isotropic reverse micelle phase under suitable conditions for the treatment. In some embodiments, nonionic surfactants are used, but are not limited thereto. Non-limiting examples of surfactants include alkylbenzene sulfonates, alkyl glycosides, alkyl sulfates, carboxylates, carboxyl esters, Cetomacrogol 1000™, octadecyl hexadecyl alcohol, hexadecyl alcohol, cocamide DEA, cocamide MEA, decyl glycosides, decyl polydextrose, disodium cocoyl diacetate, ethoxylated fatty alcohols, glyceryl monostearate, glycol esters of fatty acids, IGEPA CA-630™, isocetyl ether-20, lauryl glycosides, maltodextrin, glyceryl monolaurate, antifungalin, naphthalene sulfonate, small-range ethoxylates, and nonidet. P-40TM, Nonoxynol-9, Nonoxynol-9, NP-40TM, Octyl Glycol Dodecyl Ether, N-Octylβ-D-Thiopyranoside, Octyl Glucoside, Oleyl Alcohol, PEG-10 Sunflower Glyceryl Ester, Pentaethylene Glycol Monododecyl Ether, Polydoxylate, Poloxamer, Poloxamer 407, Polyethoxylated Tallow Amine, Polyethylene Glycol Ester, Polyglycerol Ricinol Ester, Polyoxyethylene Fatty Acid Amide, Polyoxyethylene Surfactant, Polysorbate, Polysorbate 20, Polysorbate 80, Sorbitan, Sorbitan Monolaurate, Sorbitan Monostearate, Sorbitan Tristearate, Stearyl Alcohol, Surfactant, Sulfated Alkyl Amide, Sulfonate, Triton X-100 TM Tween 80TM Skilled professionals in the relevant field will understand the elements that constitute reasonable conditions.

[0197] Conditions for antimicelle phase

[0198] After selecting a surfactant, its concentration and temperature are adjusted to form an isotropic reverse micelle phase. The surfactant concentration and temperature can be determined by referring to the surfactant's phase diagram. When a phase diagram is unavailable, it may be necessary to conduct experiments using known laboratory techniques and procedures to determine the appropriate concentration and temperature. For example, when Triton X-100... TM When used as a surfactant, a concentration of 10-60 wt% and a temperature of 40-80°C can provide a reverse micelle phase.

[0199] Sources of metal ions

[0200] One or more metal ions corresponding to the metal or alloy used for the nanoporous layer are selected for use in the liquid composition. The metal ions are added in the form of compounds containing ionic metals, such as acids, bases, or salts. Non-limiting examples of metal-derived compounds include H₂PtCl₆, H₂Pt(OH)₆, H₂PtCl₂(OH)₄, H₂Pt(SO₄)(OH)₄, PtCl₄, K₂PtCl₆, PdCl₂, and TiCl₄.

[0201] Concentration of metal ions

[0202] The concentration of metal ions was also adjusted to optimize performance. Too low a concentration prevents nanoparticle formation, while too high a concentration can affect the formation or stability of the reverse micelle phase of the surfactant. The concentrations of metal ions are approximately 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.012, 0.014, 0.016, 0.018, 0.02, 0.022, 0.024, 0.026, 0.028, 0.03, 0.032, 0.034, 0.036, 0.038, 0.04, 0.042, 0.044, 0.046, 0.048, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, or 0.1 M. In the implementation scheme, the concentration can be within a range formed by selecting any two numbers (two molar concentration values) listed in the preceding sentence, for example, between about 0.01 and about 0.03 M, between about 0.02 and 0.03 M, and so on. Within an appropriate concentration range, it has been observed that the concentration level affects the formation rate of nanoparticles.

[0203] Unlike plating bath

[0204] The reverse micelle phase prepared in step 601 is not a plating bath composition for electroplating. Unlike in a plating bath, a metal chelating agent may not be required.

[0205] Formation of nanoparticles

[0206] In step 603, the reducing agent is mixed with the aqueous liquid composition in the reverse micelle phase. When the reducing agent enters the hydrophilic core 143 of the reverse micelle 141, it reduces metal ions to metal atoms within the hydrophilic core 143. Because the metal ions are locally concentrated within the hydrophilic core 143, the metal atoms initially remain within the hydrophilic core 143. The metal atoms within each hydrophilic core 143 aggregate and grow to form metal nanoparticles. A metal nanoparticle can be grown from one reverse micelle, but is not limited to this. The resulting metal nanoparticles are generally uncharged, i.e., neutral. However, some nanoparticles may be slightly positively charged on their surface. To date, no electrical charge has been applied to form metal nanoparticles.

[0207] Nanoparticle colloids

[0208] Dispersing nanoparticles in a liquid to provide nanoparticle colloids. Figure 8 The resulting nanoparticle colloid is conceptually illustrated. During metal ion reduction and nanoparticle growth, some reverse micelles break or fracture, and thus, nanoparticles from those broken reverse micelles can disperse into a hydrophobic space. Some of these nanoparticles 151 can float freely in the resulting colloidal composition outside the hydrophilic core of the reverse micelles. Some other nanoparticles 153 can be surrounded or bound by the hydrophilic heads of surfactant molecules outside the hydrophilic core of the reverse micelles. Some nanoparticles 155 remain within the reverse micelles 141. Overall, in the resulting nanoparticle colloid, solid nanoparticles 151, 153, and 155 are dispersed in a liquid composition comprising reverse micelles 141, water, and surfactant molecules. Because nanoparticles 151, 153, and 155 are significantly separated from each other in the nanoparticle colloidal composition, the nanoparticles are unlikely to aggregate and grow into larger particles.

[0209] reducing agent

[0210] A reducing agent is a chemical entity that can donate one or more electrons to metal ions contained in nanoparticle colloids. A reducing agent is a hydrophilic compound used to enter the hydrophilic core of a reverse micelle. Non-limiting examples of hydrophilic reducing agents include ascorbic acid, acetic acid, formaldehyde, citric acid, hydroxylamine, hypophosphite, etc.

[0211] Amount of reducing agent

[0212] A hydrophilic reducing agent is added to the nanoparticle colloid in an amount sufficient to reduce the metal ions contained therein. In some embodiments, the reducing agent is added in excess, in an amount substantially greater than the stoichiometric amount of total metal ions contained in the nanoparticle colloid used for reduction. Here, "substantially greater than" means more than 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 250, 300, or 400%.

[0213] Stir

[0214] The mixture may be stirred during and / or after the addition of the reducing agent to promote its distribution. Stirring facilitates the entry of the reducing agent into the hydrophilic space of the reverse micelles. Therefore, the time required for complete reduction of metal ions in the hydrophilic space can be reduced. Stirring can be performed continuously or intermittently. In this embodiment, stirring is carried out for a duration between 1 hour and 10 hours.

[0215] Remove surfactants and form clusters

[0216] In step 605, the surfactant is substantially removed from the nanoparticle colloidal composition to form nanoparticle clusters. In the nanoparticle colloid, the surfactant stabilizes individual nanoparticles, and therefore prevents them from aggregating when a large amount of surfactant is present. To remove the surfactant from the nanoparticles, the nanoparticle colloid is subjected to centrifugation. After centrifugation, most of the nanoparticles precipitate in the bottom fraction, and surfactant molecules may be present in both the supernatant and the bottom fraction. The supernatant is separated from the bottom fraction containing the majority of the nanoparticles. In an embodiment, liquid may be added to the separated nanoparticles to dilute the surfactant in the collected bottom fraction. The liquid added to the nanoparticles may be water or an aqueous solution, and may be acidic or alkaline, but is not limited thereto. The following steps—centrifugation, collecting the bottom fraction, and adding liquid—may be repeated multiple times to collect the nanoparticles in which the surfactant has been substantially removed.

[0217] Chemical bonds between surfactants and nanoparticles

[0218] Depending on the surfactant, some nanoparticles have strong chemical bonds with the hydrophilic heads of some surfactant molecules. Surfactant molecules with negatively charged hydrophilic heads can form coordination bonds with the nanoparticle surface. Furthermore, if surfactant molecules have electron-rich hydrophilic heads (even if they are uncharged), they can also form coordination bonds with the nanoparticle surface. When using such surfactants, these chemical bonds must be broken to remove the surfactant from the nanoparticle colloid.

[0219] Breaking chemical bonds

[0220] In some implementation schemes, Figure 6B In step 604, after the formation of nanoparticles in step 603 and before centrifugation, an acidic or alkaline solution is added to the nanoparticle-surfactant colloid. The acid or alkali of the added solution causes a chemical reaction that breaks the coordination bonds between the surfactant and the nanoparticles, thereby releasing the nanoparticles. For example, protons from an acid can bond to the negatively charged or electron-rich surfactant head to release the nanoparticles. Subsequent centrifugation and collection of the bottom portion separate the nanoparticles released from the surfactant molecules. In embodiments, the addition of the acidic or alkaline solution may be performed at least once before centrifugation. In some embodiments, the addition of the acidic or alkaline solution may be performed before each centrifugation. In embodiments, the acid and alkali may be washed with water or other solvents after centrifugation.

[0221] acidic or alkaline solutions

[0222] In the embodiments, the acid or base is selected based on the surfactant to allow the surfactant molecules to be effectively separated from the nanoparticles. In the embodiments, the acidic solution has a pH value below about 3, but is not limited thereto. For example, non-limiting examples of acids used in the acidic solution include HCl, HNO3, H2SO4, HClO4, etc. In the embodiments, the alkaline solution has a pH value above about 10, but is not limited thereto. For example, non-limiting examples of bases used in the alkaline solution include NaOH, KOH, Ca(OH)2, etc.

[0223] Clustered colloids

[0224] Following or during processes used to remove surfactants and collect nanoparticles, nanoparticles tend to aggregate or condense to form nanoparticle clusters. In liquids, these clusters disperse to form cluster colloids. Each cluster comprises and is composed of metal nanoparticles that interact with each other to form a larger bulk. Individual nanoparticles within the clusters are likely to be electrically neutral. While the present invention is not bound by any theory or concept, it is believed that protons, hydroxides, and other charged electrolytes can bond to the surface of nanoparticles, and that the interaction of these electrolytes with the ions of adjacent nanoparticles can hold adjacent nanoparticles together to form clusters. In practice, the liquid cluster colloids contain a significant amount of electrolytes derived from metal ions and acidic or alkaline solutions used in the preceding preparation steps, even though surfactant molecules are largely removed.

[0225] Clusters and nanoparticles

[0226] Figure 9 TEM images of nanoparticle clusters from a diluted sample of a clustered colloid are provided. Figure 9 The two images in the text are also found in Figure 5B and 5C In these images, the clusters are irregularly shaped and range in length from approximately 30 to approximately 500 nm. The nanoparticles 121 within the clusters are discrete and generally spherical or elliptical, with a diameter of approximately 2–3 nm. Interparticle gaps 125, approximately 1–2 nm apart, exist between adjacent or neighboring nanoparticles 121. These interparticle nanopores 125 are primarily responsible for glucose oxidation in the glucose sensing electrode with the clustered nanoporous layer.

[0227] Centrifugation

[0228] Centrifugation can be performed at speeds between 3000 and 5000 rpm. Centrifugation can last between 3 and 15 minutes. After centrifugation, remove the supernatant and collect the bottom fraction containing the nanoparticles. Add liquid to the collected bottom fraction to dilute the surfactant it contains. The following steps can be repeated three times, or more: centrifugation, collecting the bottom fraction, and adding liquid.

[0229] The surfactants that are largely removed

[0230] After multiple centrifugation processes, the surfactant is largely removed. The surfactant concentration is significantly reduced in the resulting cluster colloids, but it cannot be completely removed. Initially, the reverse micelle phase contains approximately 10 to approximately 60 wt% surfactant. The resulting cluster colloids may contain no surfactant at all. In fact, the resulting cluster colloids are largely surfactant-free. The surfactant remaining in the resulting cluster colloid or in the final collection at the bottom portion may be greater than 0.0001 parts by weight per 100 parts by weight of nanoparticles and less than about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4 or 2.6 parts by weight per 100 parts by weight of nanoparticles. In the implementation scheme, the amount of the remaining surfactant, based on 100 parts by weight of nanoparticles, may be less than about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4 or 0.5 parts by weight.

[0231] Concentration of nanoparticles in clustered colloids

[0232] After multiple centrifugation processes, the total amount of nanoparticles (as part of clusters and free nanoparticles) in the final collection at the bottom portion can be approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 wt%. In embodiments, the concentration can be within a range formed by selecting any two numbers listed in the preceding sentence, for example, between approximately 20 and approximately 30 wt%, between approximately 15 and 25%, etc.

[0233] Storage cluster colloids

[0234] The clusters remain dispersed in the cluster colloid for an extended period, for example, longer than a week or a month, without any treatment. The cluster colloid can be stored in a container for a period of time after preparation and before subsequent processing. Once prepared, the cluster colloid may be available for sale and transport for processing by others or at other locations. To maintain the colloidal properties for a longer period, the concentration of nanoparticles can be adjusted after the final collection of the bottom portion. In an embodiment, the cluster colloid in the final collection of the bottom portion may be stored or transported in a container with or without concentration adjustment.

[0235] Adjust the concentration for distribution

[0236] In step 607, the collected clustered colloids can be stored for a period of time, diluted with or without a solvent. Dilution may be to adjust the concentration of clusters in the clustered colloids for subsequent processing, such as partitioning. The solvent can be water or an organic compound. One or more addition compounds may be added. Through dilution, the concentration of nanoparticles or clusters was adjusted to approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, or 15 wt%. In the implementation scheme, the concentration of nanoparticles or clusters can be within a range formed by selecting any two numbers listed in the preceding sentence, for example, between about 0.5 and about 2 wt%, between about 1 and about 3 wt%, etc. After dilution, the remaining surfactant can be less than about 0.1, 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, or 2 wt%.

[0237] Distribution cluster colloids

[0238] In step 609, the cluster colloid is dispensed onto substrate 129 to create a nanoporous layer while maintaining its colloidal properties. Various dispensing techniques can be used to dispense the cluster colloid. Dispensing can be controlled to form a dispensed cluster colloid of a certain thickness or to provide a resulting nanoporous layer of appropriate thickness after subsequent drying. Alternatively, dispensing can be controlled to provide an appropriate roughness coefficient value for the resulting nanoporous layer.

[0239] lower substrate

[0240] Cluster colloids can be applied to a substrate made of any material. In embodiments of the glucose sensing electrode, cluster colloids can be applied to a conductive or semiconducting surface of the conductive layer 110 as described above. In some embodiments, the substrate comprises two or more conductive layers.

[0241] Drying liquid to form clustered nanoporous layers

[0242] In step 611, the dispensed cluster colloid is subjected to conditions for drying a liquid. Once dispensed, the nanoparticle clusters float in the liquid and move freely in both horizontal and vertical directions. As the liquid dries, the height of the cluster colloid decreases. As the liquid continues to dry, the clusters begin to contact adjacent clusters in both the vertical and horizontal directions between the lower substrate 129 and the top of the cluster colloid. The mobility of the clusters becomes significantly restricted. After a period of time, the liquid level becomes lower than or near the top of the clusters. Once drying is complete, the nanoparticle clusters are deposited on the substrate 129, forming a nanoporous layer with a cluster morphology 120, such as... Figure 5A As shown in the image.

[0243] Thickness of nanoporous layer

[0244] The resulting nanoporous layer has the following thicknesses: approximately 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 μm. In embodiments, the thickness can be within a range formed by selecting any two numbers listed in the preceding sentence, for example, between approximately 1 μm and approximately 10 nm.

[0245] Unwashed nanoporous layer

[0246] The resulting nanoporous layer does not require washing with water or other liquids. In one embodiment, after drying, the resulting nanoporous layer, which is in a clustered morphology, is not washed with water or other liquids. In another embodiment, the nanoporous layer does not come into contact with liquids except in subsequent processing used to add a layer to the nanoporous layer.

[0247] Yield – Metal Recovery

[0248] If an excess of reducing agent is added to the nanoparticle colloid, most of the metal ions are reduced to form metal atoms, which aggregate to form nanoparticles. Subsequent treatment to remove the surfactant also collects most of the nanoparticles in clusters. Therefore, most of the metal ions added in the above process are ultimately collected as nanoparticle clusters and deposited in the resulting nanoporous layer 117. In embodiments, more than 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98% of the input metal ions are collected as nanoparticle clusters prior to distribution.

[0249] Mass production

[0250] The nanoporous layer 117 can be mass-produced by printing cluster colloids onto a substrate 129. Printing cluster colloids takes only one to two seconds. Drying liquids takes longer, but allows for greater drying space. In one embodiment, a number of individual substrates are provided, and printing can be performed on each individual substrate. Each printed substrate is then dried to form a nanoporous layer. Alternatively, multiple regions can be printed with cluster colloids on a single substrate, and the single substrate can then be cut into multiple blocks, each block including the printed regions. Individual substrates can be dried before cutting.

[0251] No electroplating or no electricity applied

[0252] Throughout the process, no electroplating was used to form the clustered morphology of the nanoporous layer. Furthermore, no electrical current was applied to the substrate 129 on which the nanoporous layer was formed.

[0253] Non-clustered nanoporous layers

[0254] Non-clustered morphology

[0255] Figure 10A A non-clustered morphology 161 of the nanoporous layer 117 is shown. As in the clustered morphology 120, the non-clustered morphology 161 includes nanoparticles 121 formed between adjacent or neighboring nanoparticles 121 and interparticle nanopores 123. The discussion of nanoparticles 121 and interparticle nanopores 123 generally applies to the non-clustered morphology 161. Figure 10BThis is a TEM image showing the non-clustered morphology of a nanoporous layer formed on a metal surface, where the darker areas represent part of the metal surface. The nanoparticles and interparticle pores in the TEM image resemble... Figure 10A Those in the diagram.

[0256] No clusters and no inter-cluster gaps

[0257] Unlike the clustered morphology 120, the non-clustered morphology 161 does not include clusters 123 or inter-cluster gaps 127. To produce the non-clustered morphology, nanoparticles are deposited on a substrate 129 by electroplating, without preparing clusters prior to electroplating. Therefore, neither clusters nor inter-cluster gaps are formed in the resulting configuration (i.e., the non-clustered morphology 161). Thus, the non-clustered morphology 161 does not possess the characteristics of the clustered morphology derived from clusters 123 or inter-cluster gaps 127.

[0258] Non-clustered cavities

[0259] When no inter-cluster gaps are present, the non-clustered morphology 161 may include internal cavities 133, which are significantly larger than the inter-particle nanopores 123. The internal cavities 133 can be formed during electroplating because nanoparticles are not always sequentially stacked on adjacent underlying surfaces. The internal cavities 133 have irregular shapes and regular sizes. The internal cavities 133 may be distributed throughout the nanoporous layer 117.

[0260] Cavities, distinct from inter-cluster gaps or spaces

[0261] The non-clustered cavity 133 differs from the inter-cluster gaps 127 of the clustered morphology 120. The cavity 133 is formed because the electroplating and deposition rates of the nanoparticles on the surface of the substrate 129 are different. The cavity 133 does not surround or define one or more clusters 125 of the nanoparticles 121. Instead, each cavity 133 is surrounded or defined by the aggregated or clustered body of the nanoparticles 121. Although the cavities 133 may be interconnected via inter-particle nanopores 123, the cavities 133 themselves do not interconnect throughout the nanoporous layer 117 or a large portion thereof. Furthermore, the cavity 133 does not occupy as much volume in the nanoporous layer 117 (which has a lower roughness coefficient in the non-clustered morphology) as the inter-cluster gaps 127 (which have a higher roughness coefficient in the clustered morphology).

[0262] Substrates largely covered with nanoparticles

[0263] See Figure 10A and 10B The top surface of substrate 129 is substantially covered by nanoparticles 121. In some embodiments, no substantial internal space is formed on or adjacent to substrate 129, but this is not a limitation.

[0264] Comparison of clustered and non-clustered morphologies

[0265] Overall, the clustered morphology 120 is much less dense than the non-clustered morphology 161. For the same thickness, the clustered morphology 120 has a higher roughness coefficient than the non-clustered morphology 161, and therefore, to achieve the same roughness coefficient, the clustered morphology 120 can be thinner than the non-clustered morphology. Also, given the irregular shape of the clusters, the inter-cluster gaps 127 of the clustered morphology 120 are typically interconnected throughout the nanoporous layer 117, unlike the internal cavities 133 of the non-clustered morphology 161 which are not connected as the inter-cluster gaps 127. Therefore, the interparticle nanopores 125 within the clusters 123 are connected to the network of inter-cluster gaps 127 in the clustered morphology 120, whereas in the non-clustered morphology 161, where inter-cluster gaps are absent, the interparticle nanopores 125 are not connected as they are in the clustered morphology 120.

[0266] Fabrication of non-clustered nanoporous layers - electroplating

[0267] The whole process

[0268] Electroplating can be used to prepare nanoporous layers with non-clustered morphologies. See also Figure 11 In step 1101, a plating bath containing metal ions and a surfactant in a reverse micelle phase is prepared. Subsequently, in step 1103, electroplating is performed in the plating bath to deposit a non-clustered nanoporous layer. In step 1105, the resulting nanoporous layer is washed to remove the surfactant.

[0269] Preparation of plating bath

[0270] In step 1101, the plating bath is similar to Figure 6A Step 601 involves the reverse micelle phase for fabricating clustered nanoporous layers without electroplating. The plating bath comprises a surfactant in the reverse micelle phase and a metal ion source material for fabricating the clustered nanoporous layers. (About...) Figure 6A All discussions of the surfactant and metal ion source materials in step 601 apply to Figure 11 Step 1101. However, the plating bath in step 1101 differs from the reverse micelle phase in step 601. An important difference may be that the plating bath may require some additional materials considering the subsequent electroplating. For many potentially spontaneously reducing metal source compounds, the plating bath may require chelating agents to prevent the spontaneous reduction of metal ions during and before electroplating. Conversely, such chelating agents may not be necessary in the reverse micelle phase of step 601.

[0271] plating

[0272] In step 1103, electroplating is performed in an aqueous liquid composition containing a reverse micelle phase of metal ions. In a plating bath containing the liquid composition, cathode and anode electrodes are immersed and connected to a power source. When a DC voltage is applied between the cathode and anode electrodes, the cathode electrode supplies electrons to the aqueous liquid composition. Electrons can jump from the cathode electrode to the nearby hydrophilic space of the reverse micelles to reduce positively charged metal ions to metal atoms within the hydrophilic space. The metal atoms aggregate and form metal particles, which can be deposited on the surface of the cathode electrode. During this process, the reverse micelles may break down. Electrons supplied to the cathode electrode pass through the deposited nanoparticles and become available on the outer surface of the deposited nanoparticles. These electrons can then be used to reduce nearby metal ions to form metal nanoparticles for deposition on the already deposited nanoparticles.

[0273] Electroplating time

[0274] Electroplating is performed for approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes to obtain a nanoporous layer with a roughness coefficient of 100 to 800. In an embodiment, the electroplating time can be within a range formed by selecting any two numbers listed in the preceding sentence, for example, between approximately 10 and approximately 30 minutes. In an embodiment, the electroplating time is controlled to obtain a nanoporous layer with a roughness coefficient of 100 or higher.

[0275] Forming layers and cavities

[0276] In the reduction process via electroplating, nanoparticles adjacent to the cathode electrode are first deposited on the cathode surface. Then, additional nanoparticles are deposited on top of the previously deposited nanoparticles 121. Thus, nanoparticles are typically deposited layer by layer on the cathode electrode. However, since the deposition of nanoparticles may not occur at the same rate across the entire cathode surface and in the previously deposited nanoparticle layers, internal cavities 133 may form within the resulting nanoporous layer. Nanoparticle deposition can occur horizontally or laterally in spaces where no nanoparticles are deposited, and some cavities 133 may be surrounded by nanoparticles formed there. While the cavities 133 may eventually interconnect via interparticle nanopores 125, micron-sized channels are not formed throughout the nanoporous layer 117 or a significant portion thereof to interconnect the cavities 133.

[0277] Surfactants deposited together

[0278] During electroplating, the reverse micelles surrounding these nanoparticles may break down, and the nanoparticles deposit on the cathode electrode. A large number of surfactant molecules from the broken reverse micelles, along with the nanoparticles, are deposited on the cathode electrode. During electroplating, surfactant molecules can bond to the surface of the nanoparticles, and nanoparticle-surfactant molecule complexes may deposit together. Surfactant molecules can insert into or be trapped between the nanoparticles in the resulting nanostructure.

[0279] Remaining surfactants and effects

[0280] The deposited surfactant molecules, along with the nanoparticles, can occupy the gaps and spaces between the nanoparticles, i.e., interparticle pores. These surfactant molecules can effectively block the nanopores and nanoparticle surfaces responsible for glucose oxidation. Furthermore, surfactant molecules can degrade on metal surfaces, potentially contaminating the nanoparticle surfaces. In summary, residual surfactants in the nanoporous layer may affect the sensitivity of glucose oxidation.

[0281] washing

[0282] In step 1105, the resulting nanoporous layer is washed with water or other liquid to remove surfactant molecules therefrom. However, because many surfactant molecules are trapped between adjacent nanoparticles, and the washing liquid may only reach a certain level, washing is not effective in removing surfactant molecules substantially.

[0283] Nanoparticle-free colloids

[0284] In the electroplating method, no reducing agent is added to reduce metal ions to form nanoparticles. During electroplating, nanoparticles can form in the hydrophilic space of reverse micelles adjacent to or near the surface of the cathode electrode. The nanoparticles may then deposit on the cathode electrode. However, nanoparticles do not form in the hydrophilic space of the reverse micelles throughout the liquid composition. Therefore, as... Figure 8 No nanoparticle colloids were formed as shown.

[0285] Cluster-free and cluster-free colloids

[0286] In this electroplating method, there is no step to remove the surfactant after the nanoparticles are formed. Instead, the surfactant and nanoparticles are deposited together during the electroplating process. Therefore, no clusters are formed at any stage of the process, nor are any cluster colloids formed.

[0287] Yield - Metal Recovery

[0288] When electroplating is complete, the plating bath contains a large number of metal ions. Therefore, the metal recovery rate in the electroplating method may not be as high as that in the process of reducing clustered nanoporous layers by adding an excess of reducing agent.

[0289] Nanoporous layers fabricated using liquid crystal phases

[0290] Nanoporous metal layers can be fabricated from liquid crystal phases of surfactants. See also... Figure 12 In step 1201, the aqueous liquid composition is prepared to contain metal ions and a surfactant in a liquid crystal phase, for example, arranged in a hexagonal pattern. Subsequently, in step 1203, the aqueous liquid composition is subjected to electroplating to deposit a nanoporous layer, wherein the liquid crystal phase is used as a template to deposit metal atoms. In step 1205, the surfactant is removed from the deposited hexagonal nanostructure. Figure 13A The formation of the hexagonal arrangement is shown. Figure 13B Metal deposition using a hexagonal arrangement of liquid crystal phases is shown.

[0291] Maltose barrier layer

[0292] maltose

[0293] Maltose is a disaccharide composed of two glucose units, such as... Figure 20 As shown. Maltose can be present in the blood or other bodily fluids of humans or animals. The presence of maltose in the test fluid can interfere with the accurate sensing of glucose levels in both enzymatic and non-enzymatic glucose sensing systems.

[0294] Maltose interference in enzyme glucose sensing

[0295] Some enzymes used in enzyme glucose sensing systems oxidize both maltose and glucose. Therefore, when maltose is present in the test fluid, the enzyme glucose sensing system can produce inaccurate glucose level readings due to the presence of maltose. Using these inaccurate readings to control or adjust insulin infusions can have serious consequences.

[0296] Maltose interference in non-enzymatic glucose sensing

[0297] The nanoporous layer 117 of the working electrode 103NE can oxidize maltose at the same bias voltage as the glucose sensing voltage. In such cases... Figure 20 At a length of approximately 1.4-1.6 nm, maltose molecules can enter the interparticle nanopores 123 of the nanoporous layer 117 and be oxidized there along with glucose. Examples 9.11 and... Figure 18 This confirmed that maltose could be detected along with glucose and other interfering chemical entities in PBS. Examples 10.9 and... Figure 19This also confirms that maltose can be detected along with glucose and other interfering chemical entities in serum.

[0298] Non-enzyme working electrode with maltose barrier layer

[0299] See Figure 21 The working electrode 103NE includes a nanoporous layer 117 and a maltose barrier or maltose screening layer 301 on the nanoporous layer 117. In an embodiment, the nanoporous layer 117 is capable of oxidizing both maltose and glucose, regardless of whether it comprises a clustered or non-clustered morphology. The maltose barrier layer 301 may be in contact with the underlying nanoporous layer 117 or may be separated by an intermediate layer. The working electrode 103NE may also include an additional functional layer 112 on the maltose barrier layer 301. Alternatively, the additional functional layer 112 may be inserted between the maltose barrier layer 301 and the nanoporous layer 117.

[0300] Selective blocking of maltose

[0301] The maltose barrier layer 301 effectively or substantially blocks or inhibits maltose molecules from passing through or penetrating it, while allowing glucose molecules to pass through. With the maltose barrier layer 301 present, maltose molecules in the test fluid cannot reach the underlying nanoporous layer 117 at a concentration that would significantly interfere with glucose sensing. Considering the selective maltose blocking effect of the maltose barrier layer 301, even if the nanoporous layer 117 is capable of oxidizing maltose at the same bias voltage as glucose oxidation, the presence of maltose in the test fluid is unlikely to affect glucose sensing. Furthermore, the maltose barrier layer 301 effectively blocks or inhibits other molecules and components larger than maltose in the test fluid.

[0302] bias voltage

[0303] In non-enzymatic glucose sensing systems, the addition of the maltose blocking layer 301 does not require increasing or decreasing the bias voltage for glucose sensing.

[0304] Porous polymer layer

[0305] In an embodiment, the maltose barrier layer 301 is made of or includes a porous polymeric material through which glucose can pass but maltose cannot. The porous polymeric material contains at least one polyphenylene diamine (poly-PD), including poly(m-phenylene diamine) (poly-mPD), poly(o-phenylene diamine) (poly-oPD), and poly(p-phenylene diamine) (poly-pPD).

[0306] Nanoscale thickness

[0307] The maltose barrier layer 301 has a thickness of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nm. Throughout this discussion, the thickness of the maltose barrier layer refers to the average thickness of the polymer layer excluding the top and bottom 10% of the thickness variation. In the implementation, the thickness can be within a range formed by selecting any two numbers (two thickness values) listed in the preceding sentence, for example, between about 15 nm and about 35 nm, between about 17 nm and about 33 nm, between about 18 nm and about 32 nm, between about 20 nm and about 30 nm, between about 21 nm and about 29 nm, between about 22 nm and about 28 nm, and so on.

[0308] Porosity level

[0309] In this embodiment, the maltose barrier layer 301 has a porosity that allows glucose molecules to pass through its thickness while effectively preventing maltose molecules from passing through. To achieve the goal of allowing glucose to pass through and blocking maltose, the total porosity of the maltose barrier layer needs to be adjusted to a desired level. The total porosity of the maltose barrier layer 301 is related to the layer's density (or internal morphology including pores and channels) and thickness. The concentration of the material used for the maltose barrier layer and the method of forming the maltose barrier layer can be related to density. While the total porosity has been successfully adjusted using these parameters, it has been found that the level of porosity is generally not definable or describable by the concentration and method of forming the layer. Although the thickness of the maltose barrier layer is also related to the total porosity, it depends on the specific porosity, or porosity per volume. Therefore, the porosity level needs to be defined in a different way.

[0310] Sensitivity (current density) to glucose and maltose without a maltose barrier layer

[0311] For glucose monitoring, under steady-state conditions with a bias voltage of 0.2–0.45 V applied to a test fluid having a glucose concentration of 4–20 mM (typical glucose levels in human body fluids), the nanoporous layer 117 (i.e., a maltose-free barrier layer) in contact with the test fluid needs to generate a voltage higher than 10 nA / mMcm. 2 The level of glucose oxidation current, 10 nA / mMcm 2This refers to the minimum current density (sensitivity) for glucose. According to the embodiment, without the maltose blocking layer, the same nanoporous layer 117 will produce a similar current level (i.e., above 10 nA / mMcm) under steady-state conditions when a bias voltage of 0.2-0.45 V is applied in a test fluid containing maltose at a concentration of 4-20 mM (the same as the glucose concentration above). 2 ).

[0312] Porosity of the maltose barrier layer determined by the current density of glucose and maltose

[0313] According to the implementation scheme, the maltose barrier layer 301 has porosity that allows glucose to move through it, such that the glucose oxidation current remains higher than the minimum current density for glucose. Therefore, when a bias voltage of 0.2-0.45V is applied in a test fluid with a glucose concentration of 4-20 mM, the working electrode 103NE with the maltose barrier layer 301 generates a current density higher than 10 nA / mMcm in steady state. 2 The level of glucose oxidation current, 10 nA / mMcm 2 This refers to the minimum current density (sensitivity) for glucose. On the other hand, the maltose barrier layer 301 has porosity that effectively prevents maltose from passing through, such that when a bias voltage of 0.2-0.45V is applied in a test fluid with a maltose concentration of 4-20 mM, the current generated solely by maltose (maltose oxidation current) in steady state is below 5 nA / mMcm. 2 At the level of 5nA / mMcm 2 This is the maximum current density for maltose when there is a maltose blocking layer.

[0314] Electrochemical polymerization

[0315] The porous polymer material used for the maltose barrier layer 301 can be formed on the nanoporous layer 117 by electrochemical polymerization (electroplation) using cyclic voltammetry. In an embodiment, a working electrode comprising the nanoporous layer is immersed in a reaction mixture solution containing monomers for cyclic voltammetric electrochemical polymerization. By applying a bias voltage within the monomer oxidation voltage range between the working electrode and the reference electrode, a polymerization reaction occurs and a polymer layer is formed on the nanoporous layer. Further details regarding the polymerization of phenylenediamine are available in the following literature: “Electropolymerization of O-Phenylenediamine on Pt-Electrode from Aqueous Acidic Solution: Kinetic, Mechanism, Electrochemical Studies and Characterization of the Polymer Obtained”, Sayyah et al., Journal of Applied Polymer Science, Vol. 112, No. 6, 3695-3706 (2009) and “Electropolymerization of P-Phenylenediamine on Pt-Electrode from Aqueous Acidic Solution: Kinetics, Mechanism, Electrochemical Studies, and Characterization of the Polymer Obtained”, Sayyah et al., Journal of Applied Polymer Science, Vol. 117, No. 2, 943-952 (2010), each of which is incorporated herein by reference.

[0316] Applying oxidation voltage

[0317] In cyclic voltammetry, the bias voltage can be varied. For example, the bias voltage can be gradually increased within the oxidation voltage range of the initial time period and then gradually decreased within the oxidation voltage range of subsequent time periods, but this is not a limitation. For phenylenediamine, a bias voltage between 0.5V and 1.0V is applied. Figure 22 An example of scanning bias voltage during cyclic voltammetric polymerization of phenylenediamine is shown.

[0318] Bias voltage scan speed

[0319] Along with the monomer concentration discussed below, the scan rate of the bias voltage between the lower and lower ends of the oxidation voltage range may be related to the porosity and thickness of the resulting polymer layer. In embodiments, the scan rate is about 0.5, 1, 2, 4, 6, 8, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 350, or 400 mV / sec. In embodiments, the scan rate may be within a range formed by selecting any two numbers listed in the preceding sentence, for example, between about 5 mV / sec and about 200 mV / sec.

[0320] Monomer concentration

[0321] The monomer concentrations are approximately 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, 9.8 or 10 mM. In the implementation scheme, the concentration of the monomer can be within a range formed by selecting any two numbers listed in the preceding sentence, for example, between about 0.05 mM and about 0.8 mM, between about 1.0 mM and about 5.0 mM, etc. The above concentrations apply to the three phenylenediamines.

[0322] Porosity considering monomer concentration

[0323] The concentration of monomer in the reaction mixture solution is related to the porosity of the resulting maltose barrier layer. Figure 24 In the flowchart for manufacturing the maltose barrier layer, the monomer concentration is first determined in step 2401 and the polymerization reaction is carried out in step 2403. In an embodiment, monomer concentrations of about 0.7 mM, about 0.6 mM, or about 0.5 mM provide the required total porosity level for the maltose barrier layer. In an embodiment, when the monomer concentration exceeds about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1.0 mM, about 1.1 mM, or about 1.2 mM, the resulting polymer layer does not have sufficient porosity to allow glucose to pass through, i.e., it produces a porosity below 10 nA / mMcm. 2 The level of glucose oxidation current, 10 nA / mMcm 2This refers to the minimum current density (sensitivity) for glucose. In step 2405, the resulting polymer layer is treated to adjust its porosity in step 2405.

[0324] Electric shock for adjusting porosity

[0325] When the total porosity of polymer layer 302 is not at the desired level, the polymer layer can be further processed to adjust the porosity. For example, the polymer layer can be subjected to electrical shock. In an embodiment, using Figure 23 The chronoamperometry setup shown allows for the application of an electric shock to the polymer layer 302, wherein the electric shock electrode 309 formed on the nanoporous layer 117 and the polymer layer 302 are immersed in an electrolyte solution 311. A power supply 305 and a switch 307 are connected between the substrate 303 and the electric shock electrode 309. With the operation of the switch 307, current flows through the porous polymer layer 302, causing a morphological change that increases the porosity of the polymer layer 302. Therefore, the polymer layer 302 becomes a maltose barrier layer 301 with a desired porosity level, allowing glucose to pass through its thickness while effectively blocking maltose from passing through.

[0326] acidic solution

[0327] The electrolyte solution used for electric shock can be an acidic solution having a pH value of about 2, 3, or 4, but is not limited thereto. In some embodiments, the acidic solution may contain at least one acid. Non-limiting examples of acids used in the acidic solution include phosphoric acid (H3PO4), nitric acid (HNO3), chloric acid (HCl), formic acid, lactic acid, malic acid, citric acid, carbonic acid, sulfonic acid, etc.

[0328] waveform of electric shock

[0329] The potential can be applied in various waveforms. In some embodiments, the potential is applied as AC or DC. In some embodiments, the potential is applied as multiple pulses or a single pulse. In some embodiments, the potential can be applied as a voltage signal of other shapes.

[0330] electric potential of electric shock

[0331] The potential applied to the polymer layer 302 is approximately or in the range of approximately 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0 V. In an embodiment, the maximum voltage may be within a range formed by selecting any two numbers listed in the preceding sentence, for example, between approximately 0.5 and approximately 2.5 V, between approximately 1.0 and approximately 2.0 V, and so on.

[0332] Duration of electric shock

[0333] The duration of the applied potential is approximately 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, or 4.5 seconds. In the implementation, the duration can be within a range formed by selecting any two numbers listed in the preceding sentence, for example, between approximately 0.5 and approximately 2.5 seconds, between approximately 1.0 and approximately 2.0 seconds, etc.

[0334] Also suitable for maltose barrier layers for enzyme sensing

[0335] In the implementation scheme, the maltose barrier layer 301 can be applied to an enzyme glucose sensing system. (See previous section) Figure 2 The maltose barrier layer 301 can be added as an additional functional layer 112 to the enzyme layer 111 to block maltose while allowing glucose to pass through.

[0336] CGM working electrode

[0337] CGM System

[0338] Continuous glucose monitoring (CGM) systems include glucose-sensing electrodes that contact a subject's biofluid in vivo to measure the glucose levels contained in the biofluid. In practice, CGM electrodes are inserted into or implanted into the subject's body to measure glucose levels over an extended period, such as days, weeks, or months.

[0339] Non-enzymatic CGM working electrode

[0340] Figure 31A cross-section of a non-enzymatic CGM working electrode 501 according to one embodiment is shown. The CGM working electrode 501 shown has a layered structure, which includes a substrate 503, a conductive layer 110, a nanoporous layer 117, a maltose barrier layer 301, an electrolyte ion barrier layer 505, and a biocompatible layer 507.

[0341] Electrode substrate

[0342] A substrate, base plate, or electrode substrate 503 provides support for the layered structure of the CGM working electrode 501. In embodiments, substrate 503 is an electrically insulating layer and may be made of or contain materials such as, but not limited to, polyimide, polypropylene, polyethylene glycol, polyhydroxyethyl methacrylate (pHEMA), and other biocompatible polymers. In embodiments, substrate 503 may be in the form of a flexible thin film of electrically insulating and biocompatible material. Substrate 503 has a thickness ranging from about 30 μm to about 200 μm, but is not limited thereto. Substrate 503 is an optional layer for the CMG sensing electrode 501 and may be omitted in some embodiments.

[0343] conductive layer

[0344] The conductive layer 110 may be disposed on the substrate 503, with or without an intermediate layer. In embodiments, the conductive layer 110 is formed by distributing a conductive or semiconducting material onto the substrate 503, but is not limited thereto. In the CGM working electrode 501, the conductive layer 110 may have a thickness ranging from about 100 nm to 100 μm, but is not limited thereto. In some embodiments, the conductive layer 119 may comprise two or more sublayers of a conductive or semiconducting material. In embodiments where the substrate 503 is omitted, the conductive layer 119 may serve as a support for a layered structure thereon.

[0345] Nanoporous layers

[0346] The nanoporous layer 117 may be formed on the conductive layer 110. In the CGM working electrode 501, the nanoporous layer 117 has a thickness ranging from about 500 nm to about 10 μm, but is not limited thereto. The nanoporous layer 117 may have at least one of the following: a clustered morphology, a non-clustered morphology, a hexagonal nanostructure, or other nanoporous morphologies.

[0347] Maltose barrier layer

[0348] A maltose barrier layer 301 may be formed on the nanoporous layer 117 to block maltose molecules from reaching the underlying nanoporous layer 117 while allowing glucose molecules to pass through. In embodiments, the maltose barrier layer 301 comprises a polymeric material such as poly-PD with nanoscale pores to allow glucose molecules to pass through while preventing maltose molecules from passing through. The maltose barrier layer may have a thickness ranging from about 5 nm to about 40 nm, but is not limited thereto. The maltose barrier layer 301 is an optional layer for the CMG sensing electrode 501 and may be omitted in some embodiments.

[0349] Electrolyte ion blocking layer (electrode modulation enhancement / promotion layer)

[0350] Electrolyte ion blocking layer 505 effectively restricts or inhibits small electrolyte ions such as Na+. + K + Ca 2+ Cl - PO4 3- and CO3 2- Diffusion occurs through or into the underlying nanoporous layer 117. As discussed later, the electrolyte ion barrier layer 505 enhances the modulation of the CGM working electrode and is also referred to as a working electrode modulation enhancement or promotion layer. The electrolyte ion barrier layer 505 is porous so that glucose molecules can freely pass through it. When implemented, the electrolyte ion barrier layer 505 is hydrophobic so that it does not rapidly expand by absorbing water contained in the test fluid. The electrolyte ion barrier layer 505 may have a thickness ranging from about 0.1 μm to about 10 μm, but is not limited to this.

[0351] Materials for electrolyte ion blocking layers

[0352] The electrolyte ion blocking layer 505 may include or be made of at least one of the following: for example, poly(methyl methacrylate) (PMMA), poly(hydroxyethyl methacrylate) (PHEMA), and poly(methyl methacrylate-co-ethylene glycol dimethacrylate) (PMMA-EG-PMMA). Furthermore, the electrolyte ion blocking layer 505 may be formed from or additionally include: a copolymer of methyl methacrylate and butyl methacrylate, and a polymer obtained by polymerization of one or more monomers, said monomers including methyl methacrylate, ethyl methacrylate, propyl methacrylate, butyl methacrylate, amyl methacrylate, hexyl methacrylate, cyclohexyl methacrylate, 2-ethylhexyl methacrylate, methyl acrylate, ethyl acrylate, propyl acrylate, butyl acrylate, amyl acrylate, hexyl acrylate, cyclohexyl acrylate, and 2-ethylhexyl acrylate.

[0353] Biocompatibility layer

[0354] When the CGM sensor is implanted or inserted into a subject, the biocompatible or bioprotective layer 507 comes into contact with the subject's tissues and bodily fluids. The biocompatible layer 507 contains at least one biocompatible material that is non-toxic to the subject's tissues and does not induce immune rejection in the subject's body. Furthermore, at least one material in the biocompatible layer 507 should allow bodily fluids to pass through it to the underlying nanoporous layer 117, so that the sensing of glucose concentration is not significantly affected by its own presence. The biocompatible layer 507 may have a thickness ranging from about 5 μM to about 30 μM, but is not limited to this.

[0355] Materials for biocompatible layers

[0356] The biocompatibility layer 507 may include or be made of at least one of the following: poly(vinyl alcohol), poly(ethylene oxide-co-propylene oxide) (PEO-PPO), poly(ethylene oxide) (PEO), poly(sulfone) (PS), poly(ethylene terephthalate) (PET), poly(ether-amine ester) (PU), poly(dimethylsiloxane) (PDMS), ethylene-co-vinyl acetate (EVA), poly(methyl methacrylate), poly(tetrafluoroethylene) (PTFE), poly(propylene) (PP), poly(ethylene) (PE), polyethylene glycol, and polyhydroxyethyl methacrylate (pHEMA).

[0357] Revise

[0358] The CGM working electrode 501 may include one or more additional functional layers, although not in Figure 31 As shown in the figure. In some embodiments, one or more of the following may be omitted: maltose barrier layer 301, electrolyte ion barrier layer 505, and biocompatibility layer 507. In other embodiments, two or more of the maltose barrier layer 301, electrolyte ion barrier layer 505, and biocompatibility layer 507 may be combined in monolayer form or their positions may be changed.

[0359] Enzyme-free layer

[0360] The CGM working electrode 501 does not include an enzyme layer containing glucose-specific enzymes. The CGM working electrode 501 does not contain any such enzymes in any of its layers.

[0361] No oxygen layer

[0362] The CGM working electrode 501 does not include the oxygen-absorbing material or layer required for collecting and supplying molecular oxygen when glucose oxidase is used for glucose oxidation.

[0363] Electron-free mediator

[0364] The CGM working electrode 501 does not include the electronic mediator required for electron transfer during glucose dehydrogenase oxidation.

[0365] Adjusting the CGM working electrode or system

[0366] Transient signal of current

[0367] After creating an electrochemical cell using a CGM working electrode under an applied bias voltage, a current is generated at the CGM working electrode. The current at the CGM working electrode is the sum of the current generated by background noise and glucose oxidation within the CGM working electrode. Initially, the current exhibits transient characteristics. Figure 25-30 As shown, initially, the current is very high compared to the current induced by glucose oxidation alone, and then decreases rapidly. Subsequently, the decay rate slows down. Eventually, the current stabilizes at a level known as steady state, although the current may fluctuate slightly within an acceptable range in vivo.

[0368] Current used for glucose sensing

[0369] For accurate glucose sensing, the current should be measured when the electrochemical cell and / or CGM working electrode are in steady state. In other words, when the glucose concentration is constant, the current from the CGM working electrode should not change too much over time (i.e., stabilize at a level after the initial drop). Furthermore, for accurate glucose sensing, the background current (noise) should not be excessively high relative to the current generated solely by glucose oxidation. In other words, the total current should not be excessively high relative to the current from glucose oxidation alone.

[0370] Adjusting the CGM working electrode or electrochemical cell

[0371] The CGM working electrode requires conditioning before glucose sensing. Conditioning refers to the process of stabilizing the CGM working electrode to ensure accurate glucose sensing. After conditioning, the current of the CGM working electrode should be stable at a level that is not excessively high relative to the current from glucose. To provide accurate glucose levels, the CGM system should use the current measured after conditioning. Conditioning the CGM working electrode can be lengthy. Commercially available enzyme CGM working electrodes require anywhere from several hours to several days to condition.

[0372] Required rate of change of current

[0373] Considering that the current generated by glucose oxidation in vivo is approximately tens of nanoamperes, for accurate glucose sensing, the decay rate of the current generated by the CGM working electrode should be less than, for example, 20 nA (nanoamperes) / min. To provide a reference point, the required rate of change of current should be at or below one of the following: 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 nA / min. In an embodiment, the rate of change of current can be determined over a shorter or longer time period.

[0374] Required current level

[0375] The current generated by the oxidation of glucose in vivo is typically in the tens of nanoamperes. The total current level required can vary due to various factors, including measurement accuracy, signal processing capabilities, and data processing capabilities. As these factors evolve, the required level may increase. Nevertheless, given that the current generated by the oxidation of glucose in vivo is approximately tens of nanoamperes, for accurate glucose sensing, the current generated from the CGM working electrode should be less than, for example, 500 nA. To provide a reference point, the required current should be at or below one of the following: 500, 490, 480, 470, 460, 450, 440, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, or 100 nA.

[0376] Adjustment complete

[0377] The CGM system determines whether the conditioning of its CGM working electrode or its electrochemical cell is complete. The CGM system can determine that conditioning is complete when the rate of change of current is or remains at or below a predetermined value (e.g., the desired rate of change or decay rate as described above). The CGM system can determine that conditioning is complete when the total current change remains at or below a predetermined value (e.g., the desired current level as described above) for a predetermined period of time. The CGM system can determine that conditioning is complete when the rate of change of current remains at or below its predetermined value, and further when the total current change remains at or below its predetermined value for a predetermined period of time (e.g., the rate of change of current is less than 5 nA / min and the total current remains less than 400 nA for 1 minute).

[0378] Notification of adjustment completed

[0379] The CGM system can notify its user of the completion of regulation. After or at some point following the formation of the electrochemical cell for glucose oxidation, the CGM system can begin monitoring the current from its CGM working electrode. When the current meets one or more regulation completion requirements, the CGM system can notify the user that regulation is complete. The notification can be issued in any form, including sound, vibration, light, or information display. Alternatively, the CGM system will not provide any information indicating glucose levels prior to regulation completion.

[0380] Reduce the settling time of the CGM working electrode

[0381] Discontinuity in the concentration of small electrolyte ions

[0382] Human body fluids contain a large number of electrolyte ions: Na+ + K + Ca 2+ Cl - PO4 3- and CO3 2- In the implementation scheme, the electrolyte ion blocking layer 505 restricts or inhibits the formation of electrolyte ions Na+. + K + Ca 2+ Cl - PO4 3- and CO3 2- They pass through it. Therefore, the concentration of these electrolyte ions is significantly different above and below the electrolyte ion blocking layer 505. Figure 32 This conceptually illustrates the concentration discontinuity on both sides of the electrolyte ion barrier layer 505. With the electrolyte ion barrier layer 505 present, the combined concentration of small electrolyte ions in the nanoporous layer 117 is significantly lower than that in the biocompatibility layer 507. Without the electrolyte ion barrier layer 505, the combined concentration of small electrolyte ions in the nanoporous layer 117 would be similar to that in the biocompatibility layer 507.

[0383] The concentration of small electrolyte ions under the electrolyte ion barrier layer

[0384] In the implementation scheme, the combined concentration of electrolyte ions below the electrolyte ion blocking layer 505 is greater than 0% of the same combined concentration of electrolyte ions above the electrolyte ion blocking layer 505, but less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20%. The combined concentration below the electrolyte ion blocking layer 505 can be within a range formed by selecting any two numbers (two % values) listed in the preceding sentence. Figure 32As shown, for example, the combined concentration of electrolyte ions in human interstitial fluid (i.e., above the electrolyte ion barrier layer 505) is about 0.1 M or higher; conversely, the combined concentration of electrolyte ions below the electrolyte ion barrier layer 505 is about 0.01 M or lower. The combined concentration of electrolyte ions below the electrolyte ion barrier layer 505 can be obtained by measuring the bilayer capacitance of the nanoporous layer 117 and substituting the measured value into the Gouy-Chapman equation, as discussed in detail in Ionic Strength-Controlled Virtual Area of ​​Mesoporous Platinum Electrode, Boo et al., J.AM.CHEM.Soc.2004, 126, 4524-4525.

[0385] Accelerated ion balance in nanoporous layers

[0386] As discussed, the ion-blocking layer 505 establishes or generates a substantial discontinuity in the combined concentration of small electrolyte ions between and below the electrolyte ion-blocking layer 505. Low concentrations of small electrolyte ions significantly outperform the regulation of the CGM working electrode 501, particularly the regulation of the nanoporous layer 117. While no aspect of the invention is bound by any theory or concept, low concentrations of small electrolyte ions can accelerate ion equilibrium in the nanoscale structure and surface of the nanoporous layer 117, which does not occur in larger-scale structures and surfaces such as micrometers. Because ion equilibrium is accelerated in the nanoporous layer 117, the time to reach ion equilibrium or steady state within the nanostructure of the nanoporous layer 117 will be shorter with lower concentrations of electrolyte ions in the presence of the electrolyte ion-blocking layer 505 than with higher concentrations without the electrolyte ion-blocking layer 505.

[0387] Significantly shortened adjustment time

[0388] With the acceleration of ion balance in the nanoporous layer 117, the electrolyte ion barrier layer 505 is significantly enhanced and promoted. Figure 31 The adjustment of the non-enzymatic CGM working electrode 501 reduces the time required to reach the desired current and / or the desired rate of change of current (i.e., steady state). According to the embodiment, when using the non-enzymatic CGM working electrode 505 with an electrolyte ion barrier layer 505, the adjustment can be completed in a fraction of the time compared to using the same non-enzymatic CGM working electrode without an electrolyte ion barrier layer 505.

[0389] Adjusting time

[0390] When the required rate of change of current is 5 nA / min or less, the non-enzymatic CGM working electrode without the electrolyte ion barrier layer 505 takes 3 hours in serum containing 0.1 M or higher electrolyte ions; in contrast, the non-enzymatic CGM working electrode with the electrolyte ion barrier layer 505 takes less than or about 1 hour 30 minutes, 1 hour 25 minutes, 1 hour 20 minutes, 1 hour 15 minutes, 1 hour 10 minutes, 1 hour 5 minutes, 1 hour, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes or 30 minutes in the same serum. When the required rate of change of current is 3 nA / min or less, the non-enzymatic CGM working electrode without the electrolyte ion barrier layer 505 consumes more than 5 hours in serum containing 0.1 M or higher electrolyte ions; conversely, the non-enzymatic CGM working electrode with the electrolyte ion barrier layer 505 consumes less than or about 1 hour 30 minutes, 1 hour 25 minutes, 1 hour 20 minutes, 1 hour 15 minutes, 1 hour 10 minutes, 1 hour 5 minutes, 1 hour, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 15 minutes, or 10 minutes in the same serum. When the required rate of change of current is 2 nA / min or less, the non-enzymatic CGM working electrode without the electrolyte ion barrier layer 505 consumes more than 5 hours or 10 hours in serum containing 0.1 M or higher electrolyte ions; conversely, the non-enzymatic CGM working electrode with the electrolyte ion barrier layer 505 consumes less than or about 1 hour 30 minutes, 1 hour 25 minutes, 1 hour 20 minutes, 1 hour 15 minutes, 1 hour 10 minutes, 1 hour 5 minutes, 1 hour, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 15 minutes or 10 minutes in the same serum.

[0391] Unexpected results

[0392] Without proper conditioning, CGM working electrodes cannot provide accurate glucose level current. Reducing conditioning time is a crucial practical issue in the development and manufacture of CGM working electrodes. This is because proper conditioning of a CGM working electrode can take several hours, if not tens of minutes, and people often want to know their glucose level immediately after insertion. Referring to the embodiments discussed later, under all other conditions being equal, by incorporating only the electrolyte ion barrier layer 505, the conditioning time of the CGM working electrode was reduced from approximately 3, 5, or 10 hours to less than 30 minutes. This is a very significant improvement and an unexpectedly high achievement.

[0393] Details of the electrolyte ion blocking layer

[0394] The electrolyte ion blocking layer 505 of the non-enzymatic CGM working electrode comprises or is made of at least one porous hydrophobic polymer, including poly(methyl methacrylate) (PMMA), poly(hydroxyethyl methacrylate) (PHEMA), and poly(methyl methacrylate-co-ethylene glycol dimethacrylate) (PMMA-EG-PMMA). Further examples of porous hydrophobic polymers include copolymers of methyl methacrylate and butyl methacrylate, and polymers obtained by polymerization of one or more monomers, including methyl methacrylate, ethyl methacrylate, propyl methacrylate, butyl methacrylate, pentyl methacrylate, hexyl methacrylate, cyclohexyl methacrylate, 2-ethylhexyl methacrylate, methyl acrylate, ethyl acrylate, propyl acrylate, butyl acrylate, pentyl acrylate, hexyl acrylate, cyclohexyl acrylate, 2-ethylhexyl acrylate, etc. These polymers have average molecular weights of approximately 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 190,000, and 200,000. The molecular weight can be any of the following values: 210,000, 220,000, 230,000, 240,000, 250,000, 260,000, 270,000, 280,000, 290,000, 300,000, 310,000, 320,000, 330,000, 340,000, 350,000, 360,000, 370,000, 380,000, 390,000, or 400,000. In this embodiment, the molecular weight can be within the range formed by selecting any two numbers listed in the preceding sentence. The electrolyte ion blocking layer may have a thickness of approximately 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 μm. In an embodiment, the thickness may be within a range formed by selecting any two numbers (two thickness values) listed in the preceding sentence, for example, between approximately 2 and approximately 5 μm, between approximately 1 and approximately 3 μm, etc.

[0395] The decrease in ion concentration had no effect on the enzyme glucose sensing electrode.

[0396] In an enzyme-based CGM system, the CGM working electrode includes a glucose-specific enzyme for oxidizing glucose molecules. The enzyme-based CGM working electrode may include a functional layer containing a porous, hydrophobic material that effectively reduces the concentration of electrolyte ions beneath the functional layer. However, in an enzyme-based CGM system, the concentration reduction achieved through the functional layer does not provide a reduction in the conditioning time of the CGM electrode, which is related to ion balance in a nanoscale surface or structure. This is because the enzyme-based CGM system uses an enzyme to oxidize glucose molecules, without requiring a nanoporous layer for glucose oxidation. Therefore, even if a porous, hydrophobic layer is included in the enzyme-based CGM working electrode, even if this layer causes a discontinuity in electrolyte ion concentration across its thickness, and even if the conditioning time of the enzyme-based CGM working electrode is reduced, this reduction is not equivalent to a reduction in conditioning time in a non-enzymatic CGM working electrode 501 that simultaneously has an electrolyte ion-blocking layer 505 and a nanoporous layer 117.

[0397] CGM Subcutaneous Electrode Module

[0398] CGM electrode unit

[0399] In one implementation, the CGM system includes an electrode unit or module for subcutaneously contracting bodily fluids in a subject. The electrode unit may include a single body housing one or more electrodes that will contact the bodily fluids when inserted into the subject's body. The single body may be flexible.

[0400] Construction of CGM electrode units

[0401] Figure 33 A CGM electrode unit 701 according to one embodiment is shown. The CGM electrode unit 701 includes a subcutaneous portion 703 and a contact terminal portion 705. The subcutaneous portion 703 is for insertion into a subject's body and includes a working electrode 501, a counter electrode 105, and a reference electrode 106, which are exposed via openings formed through an insulating layer 707 for subcutaneous contact with bodily fluids. The contact terminal portion 705 is for retention outside the subject's body and for engaging or connecting a corresponding device. The contact terminal portion 703 includes a working electrode terminal 501T, a counter electrode terminal 105T, and a reference electrode terminal 106T, which are electrically connected to the working electrode 501, counter electrode 105, and reference electrode 106 beneath the insulating layer 707, respectively. Here, the working electrode 501, counter electrode 105, and reference electrode 106 may each have the features and characteristics discussed in this disclosure, but are not limited thereto.

[0402] Manufacturing CGM electrode units

[0403] Figure 34This is a flowchart for manufacturing a CGM electrode unit 701 according to one embodiment. In step 3401, an electrically insulating flexible film is provided for the substrate or electrode substrate 503 (also... Figure 31 (in the middle). Then in step 3403, the conductive layer is as follows: Figure 35 The predetermined shapes 110R, 110W, and 110C shown are formed on substrate 503. Following this is step 3405, an insulating film 707 is applied onto the conductive layer to achieve the desired effect. Figure 36 The conductive layer is selectively exposed in portions or areas. Subsequently, in step 3407, the intermediate product is diced to provide, as shown... Figure 37 The shape shown. In step 3409, a nanoporous layer 117 is formed on the area exposed to the working electrode 501. Subsequently, in 3411, one or more functional layers are formed on the nanoporous layer 117 to provide, for example, Figure 31 The non-enzymatic CGM working electrode 501 is shown in a stacked structure. Furthermore, a salt layer can be formed on the area exposed to the reference electrode 106. In an embodiment, the intermediate product can be cleaved in step 3407 after step 3409 or 3411.

[0404] Conductive layer - multiple conductive elements

[0405] Figure 35 A top view of the intermediate product after step 3403 according to one embodiment is provided, along with a cross-section taken along line 3501 and viewed in the direction of the arrow. As shown, the conductive layer formed on the substrate 503 has three independent elements 110C, 110W, and 110R of predetermined shapes: conductive layer element 110C for the counter electrode, conductive layer element 110W for the working electrode, and conductive layer element 110R for the reference electrode. Each of the conductive layer elements 110C, 110W, and 110R includes a conductive portion reserved for contact terminals (in... Figure 33 In the contact terminal portion 705), the conductive portion reserved for the electrode (in Figure 33 (in the subcutaneous portion 703), and the conductive connection between the two conductive portions.

[0406] Manufacturing conductive layer

[0407] The conductive layer can be a single layer of conductive material or can be formed from multiple sublayers of different conductive materials. In an embodiment, either or both of the conductive layer element 110C for the counter electrode and the conductive layer element 110W for the working electrode are formed from at least two sublayers, for example, a silver layer and a conductive carbon layer on the silver layer. In an embodiment, the conductive layer element 110R for the reference electrode is formed as a single layer, such as a silver layer. The conductive layer 110 or its sublayers can be formed by printing conductive ink onto or on a substrate 503 and subsequently drying it. A sublayer formed on another sublayer can also be formed by printing conductive material onto that sublayer. Figure 35 The conductive layer elements 110W, 110C, and 110R are all monolayered; however, to demonstrate alternatives, in Figure 36-3 In 8, conductive layer elements 110W and 110C have two sublayer structures, namely a carbon layer 1605 on the silver layer 1603 (see also...). Figure 16A ).

[0408] insulating film

[0409] Figure 36 An intermediate product following the placement of an insulating film is shown according to one embodiment. The insulating film 707 can be... Figure 33 An opening is pre-cut in the subcutaneous portion 703 to expose the conductive portions reserved for the counter electrode 105, working electrode 501, and reference electrode 106. The insulating film 707 does not cover... Figure 33 The contact terminal portion 705 exposes the respective terminal portions of the conductive layer elements 110C, 110W, and 110R, which become 105T, 501T, and 106T, respectively. The conductive connections of the conductive layer elements 110C, 110W, and 110R are covered by an insulating film 707. An adhesive layer (not shown) may be inserted between the substrate film 503 and the insulating film 707. The insulating film 707 may be an adhesive-coated film.

[0410] Cutting

[0411] In step 3407, Figure 36 Intermediate products, for example, are cut by die cutting to remove excess portions of the insulating film 707 and the substrate 503. Figure 37The resulting product is shown, wherein the contact terminal portion 705 (the proximal portion of the CGM electrode unit 701) is wider than the subcutaneous portion 703 (the distal portion of the CGM electrode unit 701). In an embodiment, the distal portion has a width of approximately 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 mm in the direction along line 3501. In an embodiment, the width can be within a range formed by selecting any two numbers listed in the preceding sentence, for example, between approximately 1.0 mm and approximately 1.5 mm. In one embodiment, the CGM electrode unit 701 has a length of approximately 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 mm in the direction between its distal and proximal ends. In another embodiment, the length may be within a range formed by selecting any two numbers listed in the preceding sentence, for example, between approximately 10 mm and approximately 20 mm.

[0412] Formation of nanoporous layers

[0413] In step 3409, a nanoporous layer 117 is formed on the conductive layer element 110W exposed to the working electrode. Figure 38A The diagram shows a cross-section of the intermediate product taken along line 3501 in the direction of the arrow after the formation of the nanoporous layer 117. In one embodiment, the nanoporous layer 117 is formed by dispensing a cluster colloid containing nanoparticle clusters dispersed in a liquid onto a conductive layer 110 and allowing the liquid to dry there. Alternatively, another form of the nanoporous layer 117 can be formed using different methods disclosed herein. In some embodiments, slitting may be performed in step 3407 after the formation of the nanoporous layer 117.

[0414] Functional layer of working electrode

[0415] After forming the nanoporous layer 117, one or more functional layers are formed on the nanoporous layer 117 to provide, for example, Figure 31 The non-enzymatic CGM working electrode 501 is shown. A maltose barrier layer 301 may be formed on the nanoporous layer 117, but is not limited thereto. An electrolyte ion barrier layer 505 may be formed on the nanoporous layer 117 to improve the modulation of the generated CGM working electrode 501, but is not limited thereto. Furthermore, a biocompatibility layer 507 may be formed on the nanoporous layer 117, more specifically on the electrolyte ion barrier layer 505, but is not limited thereto. Figure 38B A cross-section of the CGM working electrode 501, including an electrolyte ion blocking layer 505 and a biocompatible layer 507, is shown.

[0416] Reference electrode and counter electrode

[0417] In one embodiment, a salt layer, such as AgCl, may be formed on the conductive layer element 110R exposed to the reference electrode 106. This salt layer can be formed at any time after the formation of the conductive layer element 110R. In another embodiment, the counter electrode 105 does not require additional treatment of the conductive layer element 110C.

[0418] Subcutaneous insertion of CGM electrode unit

[0419] In one embodiment, the subcutaneous portion 703 (distal portion) of the CGM electrode unit 701 is subcutaneously inserted into the body of a subject with or without the use of an insertion tool known in the art or to be developed thereafter. Through proper subcutaneous insertion, the working electrode 501, reference electrode 106, and counter electrode 105 of the subcutaneous portion 703 contact the interstitial fluid of the subject, while the terminal portion 705 of the CGM electrode unit 701 remains outside the subject's body.

[0420] Corresponding device

[0421] Subsequently, in an embodiment, the terminal portion 705 is engaged or connected to a corresponding device (not shown), which includes corresponding ports or terminals corresponding to the working electrode terminal 501T, the reverse electrode terminal 105T, and the reference electrode terminal 106T. In an embodiment, the corresponding device also includes circuitry that works in conjunction with the CGM electrode unit 701 for continuous glucose monitoring. Figure 1 The electrochemical cell. In some embodiments, in addition to the circuitry for completing the electrochemical cell, the corresponding device may include at least one processor for processing data, including current obtained from the electrochemical cell, to convert it into a standardized number representing glucose levels. In some embodiments, the corresponding device includes a wireless module for wirelessly transmitting data to another wireless device, such as a smartphone or computing device.

[0422] BGM disposable strip

[0423] Single-time point device

[0424] Glucose sensing can be performed in vitro at a single time point. A single-time-point glucose sensing system measures the glucose level in a test fluid (most commonly blood). Therefore, this system is called a blood glucose monitoring (BGM) system. BGM systems consist of single-use disposable cartridges or strips.

[0425] disposable boxes

[0426] Figure 39A single-timepoint glucose sensing system according to an embodiment is shown, comprising a BGM disposable cartridge 901 and a sensing module 911. The disposable cartridge 901 includes a test fluid reservoir 903, a counter electrode 105, a reference electrode 106, and a cartridge working electrode 905 formed on a substrate 907, the substrate providing structural support for electrodes 105, 106, and 905. An electrical connection (not shown) is formed between the electrodes and a connector 909 via the substrate 907.

[0427] Sensing module

[0428] In this embodiment, the disposable cartridge 901 is designed to be electrically and / or mechanically coupled to the sensing module 911 via connector 909. The sensing module 911 may include circuitry (not shown) for a voltage source 109 and a current sensor 108. When the disposable cartridge 901 is properly connected to the sensing module 911, electrodes 105, 106, and 905 are positioned in a manner similar to... Figure 1 It is connected to the circuit of the sensing module 911 in a certain way.

[0429] working electrode

[0430] According to one embodiment, the working electrode 905 includes a conductive layer 110 and a nanoporous layer 117. The working electrode 905 also includes a filter layer 913 for filtering and screening cells, lipids, and macromolecules contained in the test fluid. In one embodiment, the filter layer 913 may be made of or include woven fabric, cotton, or other materials that can screen cells, lipids, and other macromolecules in the blood while allowing glucose to pass through.

[0431] Working electrode does not include

[0432] In this embodiment, the working electrode 905 does not contain a glucose-specific enzyme. Furthermore, the working electrode 905 does not contain surfactants or electron mediators, which are essential for enzyme glucose sensing. Additionally, given that the working electrode 905 is an in vitro device, it does not require a biocompatibility layer.

[0433] Calibration of working electrode

[0434] Current from the working electrode

[0435] According to the implementation scheme, a non-enzymatic working electrode with a nanoporous glucose oxide layer generates a current caused by the oxidation of glucose contained in the test liquid. In practice, the current from the non-enzymatic working electrode includes: 1) a current generated solely by glucose oxidation (glucose oxidation current), 2) a current generated by interfering chemical entities (if contained in the test fluid), and 3) a current generated through the interaction between the electrochemical cell and other chemical entities contained in the test fluid.

[0436] glucose levels in body fluids

[0437] Normal glucose levels in healthy individuals range from 4.0 to 6.0 mM (between 72 and 108 mg / dL). For individuals with diabetes, glucose levels can range from 4.0 to 20 mM (between 72 and 360 mg / dL).

[0438] glucose oxidation current

[0439] In the implementation scheme, under steady-state conditions (after conditioning), when a bias voltage between approximately 0.2V and approximately 0.45V is applied to a test fluid containing 4.0–20 mM glucose, the current from glucose oxidation alone (glucose oxidation current) is above 10 nA / mMcm. 2 At a glucose concentration range of 4.0–20 mM, the nanoporous glucose oxide layer (and thus, the non-enzymatic working electrode) generates a glucose oxidation current of approximately 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, or 6.0 nA for 1 mM glucose in the test fluid. In an embodiment, the glucose oxidation current from 1 mM glucose in the test fluid can be within the range formed by any two numbers from the preceding sentence, for example, between 1.5 nA and 2.5 nA. Therefore, for a glucose concentration range of 4.0–20 mM, the glucose oxidation current from the non-enzymatic working electrode can be between approximately 2.0 nA (4.0 x 0.5) and approximately 120 nA (20 x 6.0). In the implementation scheme, the glucose oxidation current can be approximately 2.0, 4.0, 8.0, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, or 120Na. In the implementation scheme, the glucose oxidation current from 4.0-20 mM glucose contained in the test fluid can be within the range formed by any two numbers in the preceding sentence, for example, between about 1.5 nA and 2.5 nA.

[0440] Calibration of current and glucose concentration

[0441] In this implementation, for the same glucose concentration in the test fluid, the glucose oxidation current may differ between one nanoporous glucose oxide layer and another, depending on their specific manufacturing conditions. Furthermore, within a specific nanoporous glucose oxide layer, the glucose oxidation current is typically linearly correlated with the glucose concentration, although this may not be so linear across the entire concentration or current range. In this implementation, for each batch of nanoporous glucose oxide layers manufactured using the same conditions, one or more nanoporous glucose oxide layers are tested to determine a correlation curve between the glucose oxidation current and glucose concentration for that particular batch. Subsequently, during glucose sensing or monitoring using nanoporous glucose oxide layers from the same batch, the correlation curve is used to calculate or determine the glucose level in the test fluid.

[0442] Second working electrode

[0443] ascorbic acid

[0444] Ascorbic acid, also known as vitamin C, plays an important role in the human body. It is easily oxidized, especially at low oxidation potentials. Ascorbic acid may interfere with glucose sensing from bodily fluids.

[0445] There is currently no layer that can be used to block ascorbic acid.

[0446] Given that ascorbic acid is negatively charged, a negatively charged layer has been proposed to repel ascorbic acid as glucose passes through. However, there are currently no commercially available glucose-sensing electrodes to block ascorbic acid.

[0447] Two working electrodes

[0448] In the implementation plan, the glucose sensor or sensing system, in addition to Figure 1 In addition to the working electrode 103, there is at least one additional working electrode. Figure 40 A dual-working-electrode glucose sensing system 4101 is conceptually illustrated. In this system, a first working electrode 4103A, a second working electrode 4103B, a counter electrode 105, and a reference electrode 106 are connected to a voltage regulator 4104, which includes circuitry that functions as: operational amplifiers 4107A and 4107B, current sensors 4108A and 4108B, and voltage sources 4109A and 4109B for the two working electrodes 4103A and 4103B.

[0449] Operation of dual working electrode system

[0450] In this embodiment, the oxidation of both glucose and ascorbic acid occurs at the first working electrode 4103A. Therefore, the current from the first working electrode 4103A represents the combined concentration of glucose and ascorbic acid in the test fluid 102. On the other hand, at the second working electrode 4103B, the oxidation of ascorbic acid occurs, but the oxidation of glucose does not. Therefore, the current from the second working electrode 4103B represents only the concentration of ascorbic acid in the same test fluid 102. The difference between the two current values ​​represents the concentration or level of glucose contained in the test fluid 102.

[0451] First working electrode (glucose working electrode)

[0452] In some embodiments, the first working electrode (glucose working electrode) 4103A includes a nanoporous layer 117 on the conductive layer 110, such as Figure 3 As shown. The nanoporous layer 117 may include, but is not limited to, a clustered nanoporous structure. In other embodiments, the first working electrode 4103A may include, for example... Figure 2 The image shows an enzyme layer containing a glucose-specific enzyme for oxidizing glucose, instead of... Figure 3 The nanoporous layer 117. In any embodiment, the first working electrode 4103A does not include a negatively charged membrane or any other membrane used to inhibit ascorbic acid from passing through it.

[0453] Second working electrode (glucose-free working electrode)

[0454] The second working electrode (glucose-free working electrode) 4103B includes a conductive layer 110 but does not include any layer or feature that effectively induces glucose oxidation. In an embodiment, the second working electrode 4103B includes neither the nanoporous layer 117 nor a glucose-specific enzyme for oxidizing glucose. However, ascorbic acid oxidation occurs in the conductive layer 110. In an embodiment, the conductive layer 110 includes, but is not limited to, a conductive carbon layer formed on a silver layer.

[0455] Same bias voltage for both electrodes

[0456] In this embodiment, the same bias voltage is applied to the first working electrode 4103A and the second working electrode 4103B relative to the reference electrode 106. This is to provide an environment in which the oxidation level of ascorbic acid occurring at the first working electrode 4103A and the second working electrode 4103B is the same. Assuming that the oxidation level of ascorbic acid occurring at each of the first working electrode 4103A and the second working electrode 4103B is the same, the difference between the current from the first working electrode 4103A and the current from the second working electrode 4103B should represent glucose oxidation at the first working electrode 4103A.

[0457] Addressing interference from additional chemical entities

[0458] The dual-electrode system 4101 can be used to address interference from more than one chemical entity. In an embodiment, by adjusting the bias voltage, the first working electrode 4103A oxidizes not only glucose and ascorbic acid, but also additional interfering chemical entities, such as acetaminophen. Similarly, the second working electrode 4103B oxidizes not only ascorbic acid, but also the additional interfering chemical entities simultaneously. Here, neither the first nor the second working electrode includes any membrane for suppressing additional interfering chemical entities. The current from the first working electrode 4103A then represents the oxidation of glucose, ascorbic acid, and acetaminophen, and the current from the second working electrode 4103B represents the oxidation of ascorbic acid and acetaminophen. The difference in current represents the oxidation of glucose, which counteracts the interference from acetaminophen and ascorbic acid.

[0459] bias voltage

[0460] In the implementation, any bias voltage value in the range of 0.2–0.45V can be used to eliminate interference. In some implementations, a bias voltage value in the range of 0.2–0.32V can be used alone to address ascorbic acid interference, assuming that acetaminophen will not be oxidized in the nanoporous metal layer within this bias voltage range, which will be discussed in more detail below.

[0461] Different bias voltages

[0462] In an embodiment, the dual-electrode system 4101 may apply different bias voltages to the first and second working electrodes. For example, a first bias voltage is applied to the first working electrode 4103A, and a second bias voltage is applied to the second working electrode 4103B. Under different bias voltages, the current from the oxidation of ascorbic acid at the second working electrode 4103B may not be the same as or equal to the current component obtained from the oxidation of ascorbic acid at the first working electrode 4103A. Therefore, the current from glucose oxidation may not be a simple difference between the currents from the two electrodes. In an embodiment, however, the dual-electrode system 4101 is already implemented or connected to hardware and software to calculate an accurate glucose concentration using different bias voltages, current values ​​from the first working electrode 4103A and the second working electrode 4103B, data indicating the oxidation potential of ascorbic acid under different bias voltages, etc.

[0463] Accompanying detection

[0464] In some embodiments, the detection of the current from the first working electrode 4103A and the detection of the current from the second working electrode 4103B occur simultaneously, concurrently, in parallel, or together. In other embodiments, whether using one or two current sensors, detection can be performed at different times at specific time intervals, provided that the concentration fluctuations of the relevant chemical entity are negligible within the time interval. Those skilled in the art will understand the length of such time intervals without a significant risk of inaccuracy. For example, time intervals less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds, or time intervals less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes.

[0465] Record the concentration of interfering chemicals

[0466] In one embodiment, the dual-electrode system 4101 includes or is connected to hardware and software (not shown) configured to store current values ​​from the first working electrode 4103A and the second working electrode 4103B and / or to store the respective concentrations of glucose and ascorbic acid obtained from the current values. In some embodiments, when both ascorbic acid and acetaminophen are oxidized at the second working electrode 4103B, the hardware and software are configured to store the concentration of glucose and the combined concentration of ascorbic acid and acetaminophen.

[0467] Applicable to CGM

[0468] The dual-electrode system 4101 can be implemented in a CGM electrode unit for in vivo glucose sensing. Figure 41 A CGM electrode unit 4201 is shown, which includes a first working electrode 4103A and a second working electrode 4103B, respectively connected to the first working electrode terminal 4103AT and the second working electrode terminal 4103BT.

[0469] Suitable for background music

[0470] The dual-electrode system 4101 can be implemented in a BGM disposable kit or strip for in vitro glucose sensing. In the implementation embodiment, Figure 39 The disposable cartridge 901 may include two working electrodes. In this embodiment, the cartridge working electrode 905 serves as a first working electrode 4103A. A second working electrode 4103B may be added to the substrate 907 for contact with the test fluid. Furthermore, a corresponding sensing module 911 may include circuitry for receiving signals from the first and second working electrodes from the BGM disposable cartridge.

[0471] The first and second working electrodes must be operated together.

[0472] In the dual-electrode system 4101, two current values ​​are required: one from the first working electrode 4103A and the other from the second working electrode 4103B, in order to obtain the glucose level in the test fluid. For CGM, both the first working electrode 4103A and the second working electrode 4103B must operate continuously or repeatedly to provide the glucose level. Therefore, this system differs from any electrochemical sensing system that occasionally has a backup sensing electrode for various reasons.

[0473] Interference with acetaminophen

[0474] Acetaminophen

[0475] Acetaminophen is one of the most commonly used over-the-counter medications. Furthermore, acetaminophen is widely used as an active pharmaceutical ingredient in combination drugs.

[0476] The generally accepted problem

[0477] Given the prevalence of acetaminophen, patients who may be taking this medication also need to monitor their blood glucose levels. Considering that many glucose sensors are used by patients themselves rather than by healthcare professionals, erroneous readings caused by acetaminophen could have serious consequences. The electrochemical glucose sensing industry has recognized this problem and is seeking to address it.

[0478] There is no good solution

[0479] Numerous efforts have been made to address this issue. However, to date, no solution has convinced the industry to adopt it. There is no membrane available for the selective screening of acetaminophen to reach the electrode. Therefore, there is a long-standing unmet need.

[0480] There is no explanation for the lack of a good solution.

[0481] Commercially available electrochemical glucose sensing technology simply cannot solve this problem. This is at least partly because electrochemical glucose sensing systems are technically very complex. The working electrode has laminated components, each with its own function and independent of others. It is difficult to find a solution without affecting the function of other components and the overall performance of the working electrode. In addition to the technical complexity, developing such a product for market is also very expensive, given the industry's stringent regulatory approval process. Therefore, once a working product is approved and launched into the market, it is difficult to make significant changes to any of the working components of the approved product.

[0482] Solving the problem of non-enzymatic glucose sensing system for acetaminophen

[0483] In the implementation scheme, the non-enzymatic electrochemical glucose sensing system selectively oxidizes glucose without introducing any additional membranes for this purpose, while simultaneously not oxidizing acetaminophen. (See also...) Figure 3 and 31 The working electrode 103NE, 501 includes a conductive layer 110 and a nanoporous layer 117. The working electrode may include one or more additional functional layers on the nanoporous layer 117.

[0484] Acetaminophen-free screening membrane

[0485] In this embodiment, the working electrode 103NE does not include a membrane, thin film, or layer on the nanoporous layer 117 that is designed to selectively screen, repel, or block acetaminophen while allowing glucose to pass through. Therefore, when the working electrode 103NE comes into contact with a test fluid containing acetaminophen, both glucose and acetaminophen will come into contact with the nanoporous layer 117 and will be able to enter the nanoscale pores for oxidation therein.

[0486] Bias voltage used for oxidizing glucose and acetaminophen

[0487] In the glucose sensing system according to the embodiment, glucose is oxidized in the nanoporous layer 117 at a bias voltage between about 0.2 V and about 0.45 V. On the other hand, acetaminophen is oxidized at a bias voltage greater than 0.33, 0.34, 0.35, or 0.36 V. The bias voltage can be adjusted to induce glucose oxidation while simultaneously preventing the oxidation of acetaminophen.

[0488] Bias voltage used for selectively oxidizing glucose without oxidizing acetaminophen

[0489] In an embodiment, a bias voltage applied to the conductive layer 110 relative to the reference electrode 106 is set to induce glucose oxidation without oxidizing acetaminophen when both glucose and acetaminophen contact the nanoporous layer 117. For selective oxidation of glucose and selective non-oxidation of acetaminophen, in an embodiment, the bias voltage is set at or about 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, or 0.32 V. In an embodiment, the bias voltage can be within a range formed by selecting any two numbers (two voltage values) listed in the preceding sentence, for example, between about 0.28 V and about 0.30 V, between about 0.27 V and about 0.31 V, between about 0.26 V and about 0.30 V, between about 0.28 V and about 0.32 V, etc. In the implementation scheme, the bias voltage is below 0.30, 0.31, or 0.32V.

[0490] bias voltage in enzyme sensing electrode

[0491] For ease of comparison, a bias voltage in the range of 0.5–0.6 V is applied to the enzyme glucose sensor. In the enzyme sensing sensor, this bias voltage does not cause oxidation of glucose at its sensing electrode or elsewhere. Instead, glucose-specific enzymes oxidize glucose molecules, generating electrons to form electron mediators, which are oxidized in the conductive layer by the bias voltage. Therefore, the bias voltage causes oxidation of the electron mediators in the enzyme electrode. Example

[0492] The various aspects and features of the present invention will now be further discussed in conjunction with embodiments and experiments.

[0493] Preparation of reverse micelle phase

[0494] Example 1.1

[0495] An aqueous solution of platinum was prepared by dissolving 0.500 g (0.965 mmol) of chloroplatinic acid hexahydrate H₂PtCl₆·6H₂O (from Sigma-Aldrich) in 24.5 g of purified water under stirring. 25 g of surfactant Triton X-100 was added. TM (From Sigma-Aldrich) Added to an aqueous platinum solution to provide an aqueous composition containing a surfactant and platinum ions. The concentration of platinum ions in the aqueous composition is approximately 0.02 M. The reverse micelle phase in the aqueous composition is prepared by adjusting the temperature to 70°C with stirring.

[0496] Example 1.2

[0497] The reverse micelle phase was prepared by repeating Example 1.1, except that PtCl4·6H2O was used instead of H2PtCl6·6H2O, which could provide a platinum ion concentration of about 0.02 M in the aqueous composition.

[0498] Example 1.3

[0499] The reverse micelle phase was prepared by repeating Example 1.1, except that H2PtCl2(OH)4 was used instead of H2PtCl6·6H2O, which could provide a platinum ion concentration of about 0.02 M in the aqueous composition.

[0500] Example 1.4

[0501] The reverse micelle phase was prepared by repeating Example 1.1, except that H2Pt(SO4)(OH)4·6H2O was used instead of H2PtCl6·6H2O, which could provide a platinum ion concentration of about 0.02M in the aqueous composition.

[0502] Example 1.5

[0503] The reverse micelle phase was prepared by repeating Example 1.1, except that TiCl4·6H2O was used instead of H2PtCl6·6H2O, which could provide a titanium ion concentration of about 0.02 M in the aqueous composition.

[0504] Example 1.6

[0505] The reverse micelle phase was prepared by repeating Example 1.1, except that NP-40TM was used instead of Triton X-100 as the surfactant to provide a platinum ion concentration of about 0.02 M in the aqueous composition, and also except that the amount and temperature of the surfactant were adjusted to obtain the reverse micelle phase of the surfactant.

[0506] Example 1.7

[0507] The reverse micelle phase was prepared by repeating Example 1.1, except that polysorbate 80 was used instead of Triton X-100 as the surfactant to provide a platinum ion concentration of about 0.02 M in the aqueous composition, and also except that the amount and temperature of the surfactant were adjusted to obtain a specific surfactant reverse micelle phase.

[0508] Example 1.8

[0509] The reverse micelle phase was prepared by repeating Example 1.1, except that isocetyl ether-20 was used instead of Triton X-100 as the surfactant to provide a platinum ion concentration of about 0.02 M in the aqueous composition, and also except that the amount and temperature of the surfactant were adjusted to obtain a specific surfactant reverse micelle phase.

[0510] Example 1.9

[0511] The reverse micelle phase was prepared by repeating Example 1.1, except that poloxamer 407 was used instead of Triton X-100 as the surfactant to provide a platinum ion concentration of about 0.02 M in the aqueous composition, and also except that the amount and temperature of the surfactant were adjusted to obtain a specific surfactant reverse micelle phase.

[0512] Example 1.10

[0513] The reverse micelle phase was prepared by repeating Example 1.1, except that glycerol monolaurate was used instead of Triton X-100 as the surfactant to provide a platinum ion concentration of about 0.02 M in the aqueous composition, and also except that the amount and temperature of the surfactant were adjusted to obtain a specific surfactant reverse micelle phase.

[0514] Preparation of reducing agent

[0515] Example 2.1

[0516] An aqueous solution of the reducing agent was prepared by adding 30 g (0.170 mol) of ascorbic acid as a reducing agent to 250 ml of purified water with stirring. The reducing agent solution was heated to 70 °C. The concentration of ascorbic acid in the aqueous reducing agent solution was 0.6 M, which is equivalent to 60 times the metal ion concentration in Examples 1.1 to 1.10.

[0517] Example 2.2

[0518] The reducing agent aqueous solution was prepared by repeating Example 2.1, except that formaldehyde was used instead of ascorbic acid as the reducing agent. The amount of formaldehyde was adjusted to provide a concentration of approximately 0.6 M in the reducing agent aqueous solution.

[0519] Example 2.3

[0520] The reducing agent aqueous solution was prepared by repeating Example 2.1, except that acetic acid was used instead of ascorbic acid as the reducing agent. The amount of acetic acid was adjusted to provide a concentration of about 0.6 M in the reducing agent aqueous solution.

[0521] Example 2.4

[0522] The reducing agent aqueous solution was prepared by repeating Example 2.1, except that hypophosphite was used instead of ascorbic acid as the reducing agent. The amount of hypophosphite was adjusted to provide a concentration of approximately 0.6 M in the reducing agent aqueous solution.

[0523] Formation of nanoparticle colloids

[0524] Example 3.1

[0525] Shortly after the preparation of the reverse micelle phase, the aqueous reducing agent solution prepared in Example 2.1 was added to the aqueous composition of Example 1.1 at 70°C. In the resulting liquid composition, the concentration of platinum ions was approximately 0.0028 M, and the concentration of ascorbic acid was approximately 0.50 M. The resulting liquid composition was continuously stirred at 70°C for approximately 4 hours. A black platinum colloid was obtained.

[0526] Examples 3.2-3.10

[0527] Example 3.1 was repeated using the reverse micelle phases prepared in Examples 1.2-1.10 instead of the reverse micelle phases prepared in Example 1.1, which respectively provided the metal colloids of Examples 3.2-3.10.

[0528] Particle size analysis of nanoparticle colloids

[0529] Example 4.1

[0530] The Korea Polymer Testing Research Institute (KOPTRI) performed dynamic light scattering particle size analysis on the platinum colloid obtained in Example 3.1 using a Photal Otsuka Electronics ELS-Z2 zeta potential and particle size analyzer. For this analysis, the platinum colloid sample from Example 3.1 was dispersed at 25°C in purified water with a refractive index of 1.3328, a viscosity of 0.8878 cp, and a dielectric constant of 78.3.

[0531] Figure 14 The particle size distribution of the colloid obtained by Example 3.1 is shown. The particle size is mainly between about 9 nm and about 14 nm. This size distribution is interpreted as representing reverse micelles. The size distribution does not show diameters of 1-5 nm, which is interpreted as most platinum nanoparticles being contained or surrounded within reverse micelles. Similar results were obtained from multiple experiments according to Examples 1.1, 2.1, and 3.1.

[0532] Examples 4.2-4.10

[0533] The analysis of Example 4.1 was repeated using each colloid prepared in Examples 3.2-3.10 instead of the colloid prepared in Example 3.1. The particle size distribution of each colloid prepared in Examples 3.2-3.10 was obtained.

[0534] Remove surfactants

[0535] Example 5.1

[0536] Add 50 ml of 0.3 M HCl aqueous solution to 60 ml of platinum colloid prepared in Example 3.1. Centrifuge the acid-added platinum colloid at 3800 rpm for 10 minutes. Then discard the clear supernatant and collect the black bottom fraction. Repeat the following sequence of operations 4 times to remove the surfactant and obtain platinum colloid: add HCl aqueous solution, centrifuge, and collect the black bottom fraction.

[0537] The obtained platinum colloid was then washed with purified water to remove HCl. 50 ml of purified water was added to the collected platinum colloid. The platinum colloid with added water was centrifuged at 3800 rpm for 10 minutes. The clear supernatant was then discarded, and the black bottom fraction was collected. This process was repeated four times to remove HCl and obtain HCl-washed platinum colloid: adding purified water, centrifugation, and collecting the black bottom fraction.

[0538] Examples 5.2-5.10

[0539] Example 5.1 was repeated using the nanoparticle colloids obtained from 3.2-3.10 instead of the nanoparticle colloids prepared in Example 3.1 to collect the colloids from Examples 5.2-5.10 respectively.

[0540] Example 5.11

[0541] Example 5.1 was repeated using a 0.3M HNO3 aqueous solution instead of an HCl aqueous solution.

[0542] Example 5.12

[0543] Example 5.1 was repeated using a 0.3M NaOH aqueous solution instead of an HCl aqueous solution.

[0544] Particle size analysis of clustered colloids

[0545] Example 6.1

[0546] The Korea Polymer Testing Research Institute (KOPTRI) performed dynamic light scattering particle size analysis on the platinum colloid obtained in Example 5.1 using a Photal Otsuka Electronics ELS-Z2 zeta potential and particle size analyzer as described in Example 4.1. For this analysis, the colloidal sample from Example 5.1 was dispersed at 25°C in water with a refractive index of 1.3328, a viscosity of 0.8878 cp, and a dielectric constant of 78.3.

[0547] Figure 15 The particle size distribution of the colloid obtained by Example 5.1 is shown. The particle size is mainly between about 60 nm and about 200 nm. This size distribution is interpreted as representing irregularly shaped clusters formed by nanoparticles. Considering that the particle size in Example 4.1 is mainly between about 9 nm and about 14 nm (the size of reverse micelles rather than clusters), it should be understood that the clusters are formed by the process of Example 5.1, in which surfactant molecules are separated from the platinum nanoparticles by adding an acidic solution and the surfactant is removed by centrifugation and collection of the bottom portion. Similar results were obtained from multiple experiments according to Examples 1.1, 2.1, 3.1 and 5.1.

[0548] Examples 6.2-6.10

[0549] Example 6.1 was repeated using each colloid prepared in Examples 3.2-3.10 instead of the colloid prepared in Example 3.1. The particle size distributions of each colloid prepared in Examples 3.2-3.10 were obtained.

[0550] Platinum recovery - yield

[0551] Example 7

[0552] The cluster colloid obtained in Example 5.1 was dried. The dry weight of the colloid was 0.143 g. The colloid obtained in Example 5.1 was prepared from 60 ml of the nanoparticle colloid prepared in Example 3.1, containing 0.188 g. In this entire process, the yield of platinum was 76.1%.

[0553] Fabrication of electrodes with clustered nanoporous layers

[0554] Example 8.1 – Electrode Substrate

[0555] like Figure 16A As shown, a silver layer 1603 and a conductive carbon layer 1605 are formed on a substrate 1601 made of polyimide. The silver layer 1603 is formed by printing silver ink containing silver particles to a thickness of approximately 20 μm. The conductive carbon layer 1605 is formed by printing carbon ink containing carbon particles to a thickness of approximately 20 μm. A polyimide insulating film 1602 is laminated onto the substrate 1601 surrounding the silver layer 1603 and the conductive carbon layer 1605 to provide an electrode substrate 1606.

[0556] Example 8.2 – Formation of a nanoporous layer

[0557] The cluster colloid obtained in Example 5.1 was diluted to a concentration of 60 mg / ml. Using a microsyringe, 0.2 μL of the diluted cluster colloid was dropped onto the conductive carbon layer of electrode substrate 1606. The electrode substrate with the colloid dropped onto it was placed in an oven at 60°C for 30 minutes to form electrode 1607 comprising a platinum nanoporous layer 1609, as shown below. Figure 16B As shown.

[0558] Example 8.3 – Roughness Coefficient

[0559] The electrochemical analyzer CHI660 from CH Instruments Inc. was used as the voltage regulator 104, and the electrode 1607 prepared in Example 8.2 was used as the working electrode 103, platinum wire as the counter electrode 105, and Ag / AgCl (3M KCl) as the reference electrode 106. Figure 1 An electrochemical cell was constructed. The silver layer 1603 of electrode 1607 was connected to voltage regulator 104. 1 M H₂SO₄ aqueous solution was added instead of test fluid 102. Figure 1 In electrochemical cells.

[0560] Cyclic voltammetry was performed with a potential scan range between -0.2V and +1.2V. The actual surface area of ​​the platinum nanoporous layer was obtained by measuring the amount of protons adsorbed on its surface using cyclic voltammetry. The top surface area (geometric area) of the platinum nanoporous layer was measured. The roughness coefficient was calculated by dividing the actual surface area by the geometric area. The roughness coefficient of the nanoporous layer obtained in Example 8.2 was 1147.

[0561] Example 8.4 – Repeat Examples 8.1-8.2

[0562] Example 8.1 was repeated multiple times to prepare an additional electrode substrate. Example 8.2 was repeated multiple times using the additional electrode substrate to prepare an additional electrode 1607 comprising a platinum nanoporous layer 1609.

[0563] Example 8.5 – Repeat Example 8.3

[0564] Example 8.3 was repeated for the five electrodes 1607 prepared in Example 8.4. The roughness coefficient values ​​of the nanoporous layers were 1187, 1171, 1143, 1190, and 1119.

[0565] Example 8.6 – SEM Images

[0566] Figure 17A This is an SEM image of the top of electrode 1607 obtained from Example 8.4. The darker center indicates the region of the conductive carbon layer. Figure 17B This is a SEM image of the cross-section of electrode 1607, showing the platinum nanoporous layer 1609, carbon conductive layer 1605, and silver layer 1603 in order from top to bottom. Figure 17C Three SEM images of another electrode 1607 prepared in Example 8.4 are included. These three images were taken from above at different magnifications.

[0567] Detecting glucose in PBS

[0568] Example 9.1 – Preparation of solutions of glucose and other test materials

[0569] A 1M glucose stock solution was prepared by dissolving D-(+)-glucose powder (purchased from Sigma-Aldrich) in purified water. A 0.05M ascorbic acid (purchased from Sigma-Aldrich) was prepared by dissolving ascorbic acid (purchased from Sigma-Aldrich) in purified water. A 0.05M acetaminophen (purchased from Sigma-Aldrich) was prepared by dissolving acetaminophen (purchased from Sigma-Aldrich) in purified water. A 0.5M maltose aqueous solution was prepared by dissolving maltose (purchased from Sigma-Aldrich) in purified water.

[0570] Example 9.2 – Preparation of PBS

[0571] Prepare 500 ml of purified water containing 0.1 M NaH₂PO₄ and 0.15 M NaCl. Mix the two aqueous solutions to prepare 1 L of phosphate-buffered saline (PBS) at pH 7.4.

[0572] Example 9.3 – Preparation of a glucose sensing system in PBS

[0573] 20 ml of PBS prepared in Example 9.2 was placed in a beaker, with the temperature of the PBS maintained at 37°C. An electrochemical analyzer CHI660 from CH Instruments Inc. was used as the voltage regulator 104, and the electrode 1607 prepared in Example 8.4 was used as the working electrode 103, a platinum wire as the counter electrode 105, and Ag / AgCl (3M KCl) as the reference electrode 106. Figure 1 An electrochemical cell was constructed. The silver layer 1603 of electrode 1607 was connected to voltage regulator 104. The electrode was immersed in PBS and electrically connected to an electrochemical analyzer.

[0574] Example 9.4 – Measuring Current

[0575] In the system prepared in Example 9.3, a bias voltage of 0.4 V was applied between the working electrode 103 (electrode 1607) and the reference electrode 106. After applying the bias voltage, the current from the working electrode 103 was continuously measured. The electrochemical cell was held for 12 minutes to adjust the glucose sensing system in PBS without adding any substances. Subsequently, a current value of 0.087 μA was obtained for PBS without glucose. Figure 18 The current distribution diagrams obtained from the electrochemical cells of Examples 9.5-9.11 below are shown. Figure 18 In this context, "AA" represents ascorbic acid, and "AP" represents acetaminophen.

[0576] Example 9.5 – Sensing 1 mM glucose in PBS

[0577] After adjusting the glucose sensing system, 20 μl of the glucose stock solution prepared in Example 9.1 was added to the PBS in Example 9.3 to prepare 1 mM glucose in PBS. Immediately after addition, the PBS with added glucose was stirred for 3–4 seconds to allow the current to temporarily reach its peak. The current from the working electrode was continuously measured. Once the current stabilized, a current value of 0.54 μA was obtained for 1 mM glucose in PBS.

[0578] Example 9.6 – Sensing 3mM glucose in PBS

[0579] After the current in Example 9.5 stabilized, 40 μl of the glucose stock solution prepared in Example 9.1 was added to the PBS obtained in Example 9.4 to prepare a total of 3 mM glucose in the PBS. Immediately after addition, the PBS with added glucose was stirred for 3–4 seconds to allow the current to temporarily reach its peak. The current from the working electrode was continuously measured. Once the current stabilized, a current value of 1.19 μA was obtained for the 3 mM glucose in the PBS.

[0580] Example 9.7 – Sensing 6mM glucose in PBS

[0581] After the current in Example 9.6 stabilized, 60 μl of the glucose stock solution prepared in Example 9.1 was added to the PBS obtained in Example 9.5 to prepare a total of 6 mM glucose in the PBS. Immediately after addition, the PBS with added glucose was stirred for 3–4 seconds to allow the current to temporarily reach its peak. The current from the working electrode was continuously measured. Once the current stabilized, a current value of 2.09 μA was obtained for the 6 mM glucose in the PBS.

[0582] Example 9.8 – Sensing 10mM glucose in PBS

[0583] After the current in Example 9.7 stabilized, 80 μl of the glucose stock solution prepared in Example 9.1 was added to the PBS obtained in Example 9.6 to prepare a total of 10 mM glucose in the PBS. Immediately after addition, the PBS with added glucose was stirred for 3–4 seconds to allow the current to temporarily reach its peak. The current from the working electrode was continuously measured. Once the current stabilized, a current value of 2.89 μA was obtained for the 10 mM glucose in the PBS.

[0584] Example 9.9 – Sensing 0.11 mM ascorbic acid in PBS

[0585] After the current in Example 9.8 stabilized, 44 μl of the ascorbic acid aqueous solution prepared in Example 9.1 was added to the PBS obtained in Example 9.7 to prepare 0.11 mM ascorbic acid (AA) in PBS. Immediately after addition, the PBS with added ascorbic acid was stirred for 3–4 seconds to allow the current to temporarily reach its peak. The current from the working electrode was continuously measured. Once the current stabilized, a current value of 2.93 μA was obtained for the sum of 10 mM glucose and 0.11 mM ascorbic acid in the PBS.

[0586] Example 9.10 – Detecting 0.17 mM acetaminophen in PBS

[0587] After the current in Example 9.9 stabilized, 68 μl of the acetaminophen aqueous solution prepared in Example 9.1 was added to the PBS obtained in Example 9.8 to prepare 0.17 mM acetaminophen (AP) in PBS. Immediately after addition, the PBS with added acetaminophen was stirred for 3–4 seconds to allow the current to temporarily reach its peak. The current from the working electrode was continuously measured. Once the current stabilized, a current value of 3.21 μA was obtained for the sum of 10 mM glucose, 0.11 mM ascorbic acid, and 0.17 mM acetaminophen in the PBS.

[0588] Example 9.11 – Sensing 13.9 mM maltose in PBS

[0589] After the current in Example 9.10 stabilized, 556 μl of the maltose aqueous solution prepared in Example 9.1 was added to the PBS obtained in Example 9.9 to prepare 13.9 mM maltose in PBS. Immediately after addition, the PBS with added maltose was stirred for 3–4 seconds to allow the current to temporarily reach its peak. The current from the working electrode was continuously measured. Once the current stabilized, a current value of 4.74 μA was obtained for the sum of 10 mM glucose, 0.11 mM ascorbic acid, 0.17 mM acetaminophen, and 13.9 mM maltose in PBS.

[0590] Example 9.12 – Formula for glucose levels

[0591] In Examples 9.5-9.11, the current values ​​represent and correspond to the glucose concentration in PBS. Similar experiments were performed many times for glucose sensing systems prepared in the same manner using the same and other glucose concentrations to obtain data on current values ​​and corresponding glucose concentrations. The correlation between the glucose concentration in PBS and the current values ​​was obtained by processing the data. The glucose concentration was calculated using the correlation and current values ​​obtained from Examples 9.5-9.11.

[0592] Detecting glucose in serum

[0593] Example 10.1 – Preparation of a glucose sensing system in serum

[0594] Human serum was purchased from Sigma-Aldrich. The glucose content in the serum was measured using YSI. The serum was found to contain 5.8 mM glucose, corresponding to a blood glucose level of 104 mg / dL. 10 ml of serum was placed in a beaker, with the serum temperature maintained at 37°C. The electrochemical cell was prepared as described in Example 9.3, except that electrode 1607 prepared in Example 8.4 was used as the working electrode 103, and further except that the working electrode, reference electrode, and counter electrode were immersed in serum.

[0595] Example 10.2 – Pre-regulated serum glucose sensing system

[0596] A bias voltage of 0.4 V was applied between the working electrode 103 and the reference electrode 106 of the electrochemical cell prepared in Example 10.1. The bias voltage was maintained in the electrochemical system for more than 3 hours to regulate the system, i.e., to wait for the background current to become low enough to sense glucose oxidation. Subsequently, the bias voltage was disconnected from the system.

[0597] Example 10.3 – Measuring Current

[0598] Shortly after removing the bias voltage from Example 10.2, the same bias voltage was applied back to the system, and the current at the working electrode was measured. The electrochemical cell was held for 1.2 hours to further fine-tune the glucose sensing system in serum without adding any substances. Once the current stabilized, a current value of 96 nA was obtained for the initial 5.8 mM glucose in the serum. Figure 19 The current distribution measured from the electrochemical cells of Examples 10.4-10.9 below is shown in the diagram. Figure 19 In this context, "AA" represents ascorbic acid, and "AP" represents acetaminophen.

[0599] Example 10.4 – Detecting 10mM glucose in serum

[0600] After adjusting the glucose sensing system, 42 μl of the glucose stock solution prepared in Example 9.1 was added to the serum in Example 10.2 to prepare a total of 10 mM glucose in the serum. Immediately after addition, the glucose-added serum was stirred for 3–4 seconds to allow the current to temporarily reach its peak. The current from the working electrode was continuously measured. Once the current stabilized, a current value of 110 nA was obtained for 10 mM glucose in the serum.

[0601] Example 10.5 – Detecting 15mM glucose in serum

[0602] After the current in Example 10.4 stabilized, 50 μl of the glucose stock solution prepared in Example 9.1 was added to the serum in Example 10.3 to prepare a total of 15 mM glucose in the serum. Immediately after addition, the glucose-added serum was stirred for 3–4 seconds to allow the current to temporarily reach its peak. The current from the working electrode was continuously measured. Once the current stabilized, a current value of 132 nA was obtained for the 15 mM glucose in the serum.

[0603] Example 10.6 – Detecting 20mM glucose in serum

[0604] After the current in Example 10.5 stabilized, 50 μl of the glucose stock solution prepared in Example 9.1 was added to the serum in Example 10.4 to prepare a total of 20 mM glucose in the serum. Immediately after addition, the glucose-added serum was stirred for 3–4 seconds to allow the current to temporarily reach its peak. The current from the working electrode was continuously measured. Once the current stabilized, a current value of 159 nA was obtained for the 20 mM glucose in the serum.

[0605] Example 10.7 – Detecting 0.11 mM ascorbic acid in serum

[0606] After the current in Example 10.6 stabilized, 22 μl of the ascorbic acid aqueous solution prepared in Example 9.1 was added to the serum obtained in Example 10.5 to prepare 0.11 mM ascorbic acid (AA) in the serum. Immediately after addition, the serum with added ascorbic acid was stirred for 3–4 seconds to allow the current to temporarily reach its peak. The current from the working electrode was continuously measured. Once the current stabilized, a current value of 163 nA was obtained for the sum of 20 mM glucose and 0.11 mM ascorbic acid in the serum.

[0607] Example 10.8 – Detecting 0.17 mM acetaminophen in serum

[0608] After the current in Example 10.7 stabilized, 34 μl of the acetaminophen aqueous solution prepared in Example 9.1 was added to the serum obtained in Example 10.6 to prepare 0.17 mM acetaminophen (AP) in the serum. Immediately after addition, the serum with added acetaminophen was stirred for 3–4 seconds to allow the current to temporarily reach its peak. The current from the working electrode was continuously measured. Once the current stabilized, a current value of 223 nA was obtained for the sum of 20 mM glucose, 0.11 mM ascorbic acid, and 0.17 mM acetaminophen in the serum.

[0609] Example 10.9 – Detection of 13.9 mM maltose in serum

[0610] After the current in Example 10.8 stabilized, 278 μl of the maltose aqueous solution prepared in Example 9.1 was added to the serum obtained in Example 10.7 to prepare 13.9 mM maltose in the serum. Immediately after addition, the serum with added maltose was stirred for 3–4 seconds to allow the current to temporarily reach its peak. The current from the working electrode was continuously measured. Once the current stabilized, a current value of 231 nA was obtained for the sum of 20 mM glucose, 0.11 mM ascorbic acid, 0.17 mM acetaminophen, and 13.9 mM maltose in the serum.

[0611] Example 10.10 – Formula for glucose levels

[0612] In Examples 10.4-10.9, the current values ​​represent and correspond to the glucose concentration in serum. Similar experiments were performed many times for glucose sensing systems prepared in the same manner using the same and other glucose concentrations to obtain data on current values ​​and corresponding glucose concentrations. The correlation between serum glucose concentration and current values ​​was obtained by processing the data. Glucose concentration was calculated using the correlation and current values ​​obtained from Examples 10.4-10.9.

[0613] Non-clustered nanoporous layers

[0614] Example 11.1 – Electroplating from an inverse micelle phase

[0615] The embodiments and discussion of US Patent No. 8,343,690 ('690 Patent) are incorporated herein in their entirety. The experiments appearing in columns 6 through 9 of the '690 Patent are specifically incorporated herein as examples of fabricating nanoporous layers by electroplating and using a glucose sensing layer.

[0616] Example 11.2 – Electroplating from a Hexagonal Phase

[0617] The disclosure of US Patent No. 7,892,415 ('415 Patent) is incorporated herein in its entirety. The experiments appearing in columns 5 and 6 of the '415 Patent are specifically incorporated herein as examples of fabricating hexagonal nanoporous layers by electroplating and using a glucose sensing layer.

[0618] Example 11.3 – Electroplating from the Hexagonal Phase

[0619] The contents of “Electrochemistry Communications, Vol. 4, No. 8, August 2002, pp. 610-612” are hereby incorporated in their entirety into this document.

[0620] Example 11.4 – Chemical Deposition from Hexagonal Phase

[0621] The contents of Science, Vol. 278, October 31, 1997, pp. 838-840 are hereby incorporated in their entirety into this document.

[0622] Manufacturing a maltose barrier layer

[0623] Example 12.1 – Preparation of mPD aqueous solution

[0624] m-phenylenediamine (mPD) purchased from Sigma-Aldrich was dissolved in purified water to provide aqueous mPD solutions containing 0.1, 0.3, 0.5, 1.0, 2.0 and 5.0 mM mPD.

[0625] Example 12.2 – Preparing for Cyclic Voltammetry

[0626] An electrochemical cell was prepared using a CHI Multi 1030C electrochemical analyzer from CH Instruments Inc. as the voltage regulator 104, and using the electrode 1607 prepared in Example 8.4 as the working electrode 103, a platinum wire as the counter electrode 105, and Ag / AgCl (3MKCl) as the reference electrode 106. The counter electrode 105 and the reference electrode 106 were electrically connected to form a two-electrode system.

[0627] Example 12.3 – Electrochemical polymerization at 0.1 mM and 10 mV / s

[0628] In the electrochemical cell prepared in Example 12.2, a 0.1 mM mPD aqueous solution prepared in Example 12.1 was added to replace the test fluid 102. Figure 22 As shown, cyclic voltammetry was performed on two scanning segments at a scan rate of 10 mV / s using a potential scan range between +0.5 V and +1.0 V, thereby generating a poly-mPD maltose barrier layer 301 on the nanoporous layer 117.

[0629] Example 12.4 – Electrochemical polymerization at 0.1 mM and 100 mV / s

[0630] Example 12.3 was repeated, except that the scan rate was 100 mV / s, which formed a poly-mPD layer on the nanoporous layer 117.

[0631] Example 12.5 – Electrochemical polymerization at 0.1 mM and 200 mV / s

[0632] Example 12.3 was repeated, except that the scan rate was 200 mV / s, which formed a poly-mPD layer on the nanoporous layer 117.

[0633] Example 12.6 – Electrochemical polymerization at 0.3 mM and 10 mV / s

[0634] Example 12.3 was repeated, except that the 0.3 mM mPD aqueous solution prepared in Example 12.1 was added instead of the 0.1 mM mPD aqueous solution, which formed a poly-mPD layer on the nanoporous layer 117.

[0635] Example 12.7 – Electrochemical polymerization at 0.3 mM and 100 mV / s

[0636] Example 12.6 was repeated, except that the scan rate was 100 mV / s, which formed a poly-mPD layer on the nanoporous layer 117.

[0637] Example 12.8 – Electrochemical polymerization at 0.3 mM and 200 mV / s

[0638] Example 12.6 was repeated, except that the scan rate was 200 mV / s, which formed a poly-mPD layer on the nanoporous layer 117.

[0639] Example 12.9 – Electrochemical polymerization at 0.5 mM and 10 mV / s

[0640] Example 12.3 was repeated, except that the 0.5 mM mPD aqueous solution prepared in Example 12.1 was added instead of the 0.1 mM mPD aqueous solution, which formed a poly-mPD layer on the nanoporous layer 117.

[0641] Example 12.10 – Electrochemical polymerization at 0.5 mM and 100 mV / s

[0642] Example 12.6 was repeated, except that the scan rate was 100 mV / s, which formed a poly-mPD layer on the nanoporous layer 117.

[0643] Example 12.11 – Electrochemical polymerization at 0.5 mM and 200 mV / s

[0644] Example 12.6 was repeated, except that the scan rate was 200 mV / s, which formed a poly-mPD layer on the nanoporous layer 117.

[0645] Example 12.12 – Electrochemical polymerization at 1.0 mM and 10 mV / s

[0646] Example 12.3 was repeated, except that a 1.0 mM mPD aqueous solution prepared in Example 12.1 was added instead of a 0.1 mM mPD aqueous solution, which formed a poly-mPD layer on the nanoporous layer 117.

[0647] Example 12.13 – Electric Shock

[0648] The poly-mPD layer prepared in Example 12.12 was used as the porous polymer layer, and 302 and 1M H2SO4 aqueous solution were used as the electrolyte solution for the preparation of chronoamperometry. Figure 23 An electrochemical cell. An electric shock is applied to the porous polymer layer 302 by applying a single pulse with a pulse width of 1.0 second, ranging from +0.0V to +1.0V.

[0649] Example 12.14 – Electrochemical polymerization at 1.0 mM, 100 mV / s and electric shock

[0650] Example 12.6 was repeated, except that the scan rate was 100 mV / s, which formed a poly-mPD layer on the nanoporous layer 117. Subsequently, Example 12.13 was repeated using the poly-mPD layer formed on the nanoporous layer.

[0651] Example 12.15 – Electrochemical polymerization at 1.0 mM, 200 mV / s and under electric shock

[0652] Example 12.6 was repeated, except that the scan rate was 200 mV / s, which formed a poly-mPD layer on the nanoporous layer 117. Subsequently, Example 12.13 was repeated using the poly-mPD layer formed on the nanoporous layer.

[0653] Example 12.16 – Electrochemical polymerization at 2.0 mM, 10 mV / s and electric shock

[0654] Example 12.3 was repeated, except that the 2.0 mM mPD aqueous solution prepared in Example 12.1 was added instead of the 0.1 mM mPD aqueous solution, which formed a poly-mPD layer on the nanoporous layer 117. Subsequently, Example 12.13 was repeated using the poly-mPD layer formed on the nanoporous layer.

[0655] Example 12.17 – Electrochemical polymerization at 2.0 mM, 100 mV / s and under electric shock

[0656] Example 12.6 was repeated, except that the scan rate was 100 mV / s, which formed a poly-mPD layer on the nanoporous layer 117. Subsequently, Example 12.13 was repeated using the poly-mPD layer formed on the nanoporous layer.

[0657] Example 12.18 – Electrochemical polymerization at 2.0 mM, 200 mV / s and electric shock

[0658] Example 12.6 was repeated, except that the scan rate was 200 mV / s, which formed a poly-mPD layer on the nanoporous layer 117. Subsequently, Example 12.13 was repeated using the poly-mPD layer formed on the nanoporous layer.

[0659] Example 12.19 – Electrochemical polymerization at 5.0 mM, 10 mV / s and electric shock

[0660] Example 12.3 was repeated, except that the 5.0 mM mPD aqueous solution prepared in Example 12.1 was added instead of the 0.1 mM mPD aqueous solution, which formed a poly-mPD layer on the nanoporous layer 117. Subsequently, Example 12.13 was repeated using the poly-mPD layer formed on the nanoporous layer.

[0661] Example 12.20 – Electrochemical polymerization at 5.0 mM, 100 mV / s and electric shock

[0662] Example 12.6 was repeated, except that the scan rate was 100 mV / s, which formed a poly-mPD layer on the nanoporous layer 117. Subsequently, Example 12.13 was repeated using the poly-mPD layer formed on the nanoporous layer.

[0663] Example 12.21 – Electrochemical polymerization at 5.0 mM, 200 mV / s and electric shock

[0664] Example 12.6 was repeated, except that the scan rate was 200 mV / s, which formed a poly-mPD layer on the nanoporous layer 117. Subsequently, Example 12.13 was repeated using the poly-mPD layer formed on the nanoporous layer.

[0665] Detecting glucose without maltose interference

[0666] Example 13.1 – Preparation of serum

[0667] Human serum was purchased from Sigma-Aldrich. Glucose levels in the serum were measured using YSI. The serum was found to contain 5.8 mM glucose, corresponding to a blood glucose level of 104 mg / dL.

[0668] Example 13.2 – Preparation of a glucose sensing system in serum

[0669] 10 ml of serum prepared in Example 13.1 was placed in a beaker, with the serum temperature maintained at 37°C. An electrochemical cell was prepared as described in Example 10.2, except that the working electrode 103 included a poly-mPD maltose barrier layer 301 on a nanoporous layer, which was prepared using a 0.1 mM mPD solution and a scan rate of 10 mV / second as in Example 12.3.

[0670] Example 13.1 – Preparation of a glucose sensing system in serum

[0671] The electrochemical cell was prepared by repeating Example 10.2, except that the working electrode 103 included a poly-mPD maltose barrier layer 301 on a nanoporous layer prepared as in Example 12.3 (using 0.1 mM mPD solution and a scan rate of 10 mV / s), and also except that the working electrode, reference electrode and counter electrode were immersed in serum.

[0672] Example 13.2 – Regulation of Glucose Sensing Systems in Serum

[0673] In the electrochemical cell system prepared in Example 13.1, a bias voltage of 0.4 V was applied between the working electrode 103 and the reference electrode 106. The bias voltage was maintained in the electrochemical system for more than 3 hours to pre-adjust the system. Subsequently, the bias voltage was disconnected from the system and reconnected. After the bias voltage was reapplied, the current from the working electrode was measured. The electrochemical cell was retained to further adjust the glucose sensing system in serum. Once the current stabilized, a current value of 96 nA was measured for the initial 5.8 mM glucose in the serum.

[0674] Example 13.3 – Electrode with maltose barrier layer (0.1mM, 10mV / sec)

[0675] In the system prepared in Example 13.2, the glucose stock solution prepared in Example 9.1 was added to serum to obtain a total glucose concentration of 10 mM in serum. Subsequently, the glucose stock solution was further added to obtain total glucose concentrations of 15 mM and 20 mM in serum, with time intervals between each addition. Then, the ascorbic acid aqueous solution prepared in Example 9.1 was added to serum to obtain 0.11 mM ascorbic acid in serum. Next, the acetaminophen aqueous solution prepared in Example 9.1 was added to the resulting serum to obtain 0.17 mM acetaminophen in serum. Then, the maltose aqueous solution prepared in Example 9.1 was added to the resulting serum to obtain 13.9 mM maltose in serum. After each addition, the serum was immediately stirred for 3-4 seconds to allow the current to temporarily reach its peak value. Figure 25 The monitored current in this embodiment is shown in red. Changes in current were observed with each addition of glucose, ascorbic acid (AA), and acetaminophen (AP). However, after the addition of maltose, a change in current of no more than 5 nA / mMcm was observed. 2 In addition to the peak caused by stirring, in this embodiment, the maltose barrier layer effectively blocks maltose without interrupting glucose sensing.

[0676] Example 13.4 – Electrode with maltose barrier layer (0.1mM, 100mV / sec)

[0677] Examples 13.1-13.3 are repeated, except that the working electrode 103 includes a maltose barrier layer prepared as in Example 12.4 (using 0.1 mM mPD solution at a scan rate of 100 mV / sec). Figure 25 The monitored current in this embodiment is shown in green. Changes in current were observed with each addition of glucose, ascorbic acid, and acetaminophen. However, after the addition of maltose, a change in current of no more than 5 nA / mMcm was observed. 2 In addition to the peak caused by stirring, in this embodiment, the maltose barrier layer effectively blocks maltose without interrupting glucose sensing.

[0678] Example 13.5 – Electrode with maltose barrier layer (0.1mM, 200mV / sec)

[0679] Examples 13.1-13.3 are repeated, except that the working electrode 103 includes a maltose barrier layer prepared as in Example 12.5 (using 0.1 mM mPD solution at a scan rate of 200 mV / sec). Figure 25 The monitored current in this embodiment is shown in purple. Changes in current were observed with each addition of glucose, ascorbic acid, and acetaminophen. However, after the addition of maltose, a change in current of no more than 5 nA / mMcm was observed. 2In addition to the peak caused by stirring, in this embodiment, the maltose barrier layer effectively blocks maltose without interrupting glucose sensing.

[0680] Example 13.6 – Electrode with maltose barrier layer (0.3mM, 10mV / sec)

[0681] Examples 13.1-13.3 are repeated, except that the working electrode comprises a maltose barrier layer prepared as in Example 12.6 (using 0.3 mM mPD solution at a scan rate of 10 mV / sec). Figure 26 The monitored current in this embodiment is shown in red. Changes in current were observed with each addition of glucose, ascorbic acid, and acetaminophen. However, after the addition of maltose, a change in current of no more than 5 nA / mMcm was observed. 2 In addition to the peak caused by stirring, in this embodiment, the maltose barrier layer effectively blocks maltose without interrupting glucose sensing.

[0682] Example 13.7 – Electrode with maltose barrier layer (0.3mM, 100mV / sec)

[0683] Examples 13.1-13.3 are repeated, except that the working electrode comprises a maltose barrier layer prepared as in Example 12.7 (using 0.3 mM mPD solution at a scan rate of 100 mV / sec). Figure 26 The monitored current in this embodiment is shown in green. Changes in current were observed with each addition of glucose, ascorbic acid, and acetaminophen. However, after the addition of maltose, a change in current of no more than 5 nA / mMcm was observed. 2 In addition to the peak caused by stirring, in this embodiment, the maltose barrier layer effectively blocks maltose without interrupting glucose sensing.

[0684] Example 13.8 – Electrode with maltose barrier layer (0.3mM, 200mV / sec)

[0685] Examples 13.1-13.3 are repeated, except that the working electrode comprises a maltose barrier layer prepared as in Example 12.8 (using 0.3 mM mPD solution at a scan rate of 200 mV / sec). Figure 26 The monitored current in this embodiment is shown in purple. Changes in current were observed with each addition of glucose, ascorbic acid, and acetaminophen. However, after the addition of maltose, a change in current of no more than 5 nA / mMcm was observed. 2 In addition to the peak caused by stirring, in this embodiment, the maltose barrier layer effectively blocks maltose without interrupting glucose sensing.

[0686] Example 13.9 – Electrode with maltose barrier layer (0.5mM, 10mV / sec)

[0687] Examples 13.1-13.3 are repeated, except that the working electrode comprises a maltose barrier layer prepared as in Example 12.9 (using 0.5 mM mPD solution at a scan rate of 10 mV / sec). Figure 27 The monitored current in this embodiment is shown in red. Changes in current were observed with each addition of glucose, ascorbic acid, and acetaminophen. However, after the addition of maltose, a change in current of no more than 5 nA / mMcm was observed. 2 In addition to the peak caused by stirring, in this embodiment, the maltose barrier layer effectively blocks maltose without interrupting glucose sensing.

[0688] Example 13.10 – Electrode with maltose barrier layer (0.5mM, 100mV / sec)

[0689] Examples 13.1-13.3 are repeated, except that the working electrode comprises a maltose barrier layer prepared as in Example 12.9 (using 0.5 mM mPD solution at a scan rate of 100 mV / sec). Figure 27 The monitored current in this embodiment is shown in green. Changes in current were observed with each addition of glucose, ascorbic acid, and acetaminophen. However, after the addition of maltose, a change in current of no more than 5 nA / mMcm was observed. 2 In addition to the peak caused by stirring, in this embodiment, the maltose barrier layer effectively blocks maltose without interrupting glucose sensing.

[0690] Example 13.11 – Electrode with maltose barrier layer (0.5mM, 200mV / sec)

[0691] Examples 13.1-13.3 are repeated, except that the working electrode comprises a maltose barrier layer prepared as in Example 12.11 (using 0.5 mM mPD solution at a scan rate of 200 mV / sec). Figure 27 The monitored current in this embodiment is shown in purple. Changes in current were observed with each addition of glucose, ascorbic acid, and acetaminophen. However, after the addition of maltose, a change in current of no more than 5 nA / mMcm was observed. 2 In addition to the peak caused by stirring, in this embodiment, the maltose barrier layer effectively blocks maltose without interrupting glucose sensing.

[0692] Example 13.12 – Electrode with maltose barrier layer (1.0 mM, 10 mV / s)

[0693] Examples 13.1-13.3 are repeated, except that the working electrode comprises a maltose barrier layer prepared as in Example 12.12 (using 1.0 mM mPD solution at a scan rate of 10 mV / sec) and is further subjected to the electric shock as in Example 12.13. Figure 28The monitored current in this embodiment is shown in red. Changes in current were observed with each addition of glucose, ascorbic acid, and acetaminophen. However, after the addition of maltose, a change in current of no more than 5 nA / mMcm was observed. 2 In addition to the peak caused by stirring, in this embodiment, the maltose barrier layer effectively blocks maltose without interrupting glucose sensing.

[0694] Example 13.13 – Electrode with maltose barrier layer (1.0 mM, 100 mV / s)

[0695] Examples 13.1-13.3 are repeated, except that the working electrode comprises a maltose barrier layer prepared as in Example 12.14 (using 1.0 mM mPD solution at a scan rate of 100 mV / sec) and is further subjected to an electric shock as in Example 12.13. Figure 28 The monitored current in this embodiment is shown in green. Changes in current were observed with each addition of glucose, ascorbic acid, and acetaminophen. However, after the addition of maltose, a change in current of no more than 5 nA / mMcm was observed. 2 In addition to the peak caused by stirring, in this embodiment, the maltose barrier layer effectively blocks maltose without interrupting glucose sensing.

[0696] Example 13.14 – Electrode with maltose barrier layer (1.0 mM, 200 mV / s)

[0697] Examples 13.1-13.3 are repeated, except that the working electrode comprises a maltose barrier layer prepared as in Example 12.15 (using 1.0 mM mPD solution at a scan rate of 200 mV / sec) and is further subjected to electric shock. Figure 28 The monitored current in this embodiment is shown in purple. Changes in current were observed with each addition of glucose, ascorbic acid, and acetaminophen. However, after the addition of maltose, a change in current of no more than 5 nA / mMcm was observed. 2 In addition to the peak caused by stirring, in this embodiment, the maltose barrier layer effectively blocks maltose without interrupting glucose sensing.

[0698] Example 13.15 – Electrode with maltose barrier layer (2.0 mM, 10 mV / s)

[0699] Examples 13.1-13.3 are repeated, except that the working electrode comprises a maltose barrier layer prepared as in Example 12.16 (using 2.0 mM mPD solution at a scan rate of 10 mV / sec) and is further subjected to an electric shock as in Example 12.15. Figure 29The monitored current in this embodiment is shown in red. Changes in current were observed with each addition of glucose, ascorbic acid, and acetaminophen. However, after the addition of maltose, a change in current of no more than 5 nA / mMcm was observed. 2 In addition to the peak caused by stirring, in this embodiment, the maltose barrier layer effectively blocks maltose without interrupting glucose sensing.

[0700] Example 13.16 – Electrode with maltose barrier layer (2.0 mM, 100 mV / s)

[0701] Examples 13.1-13.3 are repeated, except that the working electrode comprises a maltose barrier layer prepared as in Example 12.17 (using 2.0 mM mPD solution at a scan rate of 100 mV / sec) and is further subjected to an electric shock as in Example 12.15. Figure 29 The monitored current in this embodiment is shown in green. Changes in current were observed with each addition of glucose, ascorbic acid, and acetaminophen. However, after the addition of maltose, a change in current of no more than 5 nA / mMcm was observed. 2 In addition to the peak caused by stirring, in this embodiment, the maltose barrier layer effectively blocks maltose without interrupting glucose sensing.

[0702] Example 13.17 – Electrode with maltose barrier layer (2.0 mM, 200 mV / s)

[0703] Examples 13.1-13.3 are repeated, except that the working electrode comprises a maltose barrier layer prepared as in Example 12.18 (using 2.0 mM mPD solution at a scan rate of 200 mV / sec) and is further subjected to an electric shock as in Example 12.15. Figure 29 The monitored current in this embodiment is shown in purple. Changes in current were observed with each addition of glucose, ascorbic acid, and acetaminophen. However, after the addition of maltose, a change in current of no more than 5 nA / mMcm was observed. 2 In addition to the peak caused by stirring, in this embodiment, the maltose barrier layer effectively blocks maltose without interrupting glucose sensing.

[0704] Example 13.18 – Electrode with maltose barrier layer (5.0 mM, 10 mV / s)

[0705] Examples 13.1-13.3 are repeated, except that the working electrode comprises a maltose barrier layer prepared as in Example 12.19 (using 5.0 mM mPD solution at a scan rate of 10 mV / sec) and is further subjected to an electric shock as in Example 12.15. Figure 30The monitored current in this embodiment is shown in red. Changes in current were observed with each addition of glucose, ascorbic acid, and acetaminophen. However, after the addition of maltose, a change in current of no more than 5 nA / mMcm was observed. 2 In addition to the peak caused by stirring, in this embodiment, the maltose barrier layer effectively blocks maltose without interrupting glucose sensing.

[0706] Example 13.19 – Electrode with maltose barrier layer (5.0 mM, 100 mV / s)

[0707] Examples 13.1-13.3 are repeated, except that the working electrode comprises a maltose barrier layer prepared as in Example 12.20 (using 5.0 mM mPD solution at a scan rate of 100 mV / sec) and is further subjected to an electric shock as in Example 12.15. Figure 30 The monitored current in this embodiment is shown in green. Changes in current were observed with each addition of glucose, ascorbic acid, and acetaminophen. However, after the addition of maltose, a change in current of no more than 5 nA / mMcm was observed. 2 In addition to the peak caused by stirring, in this embodiment, the maltose barrier layer effectively blocks maltose without interrupting glucose sensing.

[0708] Example 13.20 – Electrode with maltose barrier layer (5.0 mM, 200 mV / s)

[0709] Examples 13.1-13.3 are repeated, except that the working electrode comprises a maltose barrier layer prepared as in Example 12.21 (using 5.0 mM mPD solution at a scan rate of 200 mV / sec) and is further subjected to an electric shock as in Example 12.15. Figure 30 The monitored current in this embodiment is shown in purple. Changes in current were observed with each addition of glucose, ascorbic acid, and acetaminophen. However, after the addition of maltose, a change in current of no more than 5 nA / mMcm was observed. 2 In addition to the peak caused by stirring, in this embodiment, the maltose barrier layer effectively blocks maltose without interrupting glucose sensing.

[0710] Example 13.21 – Electrode with maltose barrier layer (1.0 mM, 10 mV / s)

[0711] Example 13.12 was repeated, except that the poly-mPD layer prepared in Example 12.12 (using a 1.0 mM mPD solution at a scan rate of 10 mV / sec) was not subjected to electric shock.

[0712] Example 13.22 – Electrode with maltose barrier layer (1.0 mM, 100 mV / s)

[0713] Example 13.12 was repeated, except that the poly-mPD layer prepared in Example 12.14 (using a 1.0 mM mPD solution at a scan rate of 100 mV / sec) was not subject to electric shock.

[0714] Example 13.23 – Electrode with maltose barrier layer (1.0 mM, 200 mV / s)

[0715] Example 13.12 was repeated, except that the poly-mPD layer prepared in Example 12.15 (using a 1.0 mM mPD solution at a scan rate of 200 mV / sec) was not subjected to electric shock.

[0716] Example 13.24 – Electrode with maltose barrier layer (2.0 mM, 10 mV / s)

[0717] Example 13.12 was repeated, except that the poly-mPD layer prepared in Example 12.16 (using a 2.0 mM mPD solution at a scan rate of 10 mV / sec) was not subjected to electric shock. No change in current was observed in response to each addition of glucose, which means that the poly-mPD layer effectively blocked glucose.

[0718] Example 13.25 – Electrode with maltose barrier layer (2.0 mM, 100 mV / s)

[0719] Example 13.12 was repeated, except that the poly-mPD layer prepared in Example 12.17 (using a 2.0 mM mPD solution at a scan rate of 100 mV / sec) was not subjected to electric shock. No change in current was observed in response to each addition of glucose, which means that the poly-mPD layer effectively blocked glucose.

[0720] Example 13.26 – Electrode with maltose barrier layer (2.0 mM, 200 mV / s)

[0721] Example 13.12 was repeated, except that the poly-mPD layer prepared in Example 12.18 (using a 2.0 mM mPD solution at a scan rate of 200 mV / sec) was not subjected to electric shock. No change in current was observed in response to each addition of glucose, which means that the poly-mPD layer effectively blocked glucose.

[0722] Example 13.27 – Electrode with maltose barrier layer (5.0 mM, 10 mV / s)

[0723] Example 13.12 was repeated, except that the poly-mPD layer prepared in Example 12.19 (using a 5.0 mM mPD solution at a scan rate of 10 mV / sec) was not subjected to electric shock. No change in current was observed in response to each addition of glucose, which means that the poly-mPD layer effectively blocked glucose.

[0724] Example 13.28 – Electrode with maltose barrier layer (5.0 mM, 100 mV / s)

[0725] Example 13.12 was repeated, except that the poly-mPD layer prepared in Example 12.20 (using a 5.0 mM mPD solution at a scan rate of 100 mV / sec) was not subjected to electric shock. No change in current was observed in response to each addition of glucose, which means that the poly-mPD layer effectively blocked glucose.

[0726] Example 13.29 – Electrode with maltose barrier layer (5.0 mM, 200 mV / s)

[0727] Example 13.12 was repeated, except that the poly-mPD layer prepared in Example 12.21 (using a 5.0 mM mPD solution at a scan rate of 200 mV / sec) was not subjected to electric shock. No change in current was observed in response to each addition of glucose, which means that the poly-mPD layer effectively blocked glucose.

[0728] Alternative electric shock

[0729] Example 14.1 – Two-pulse electric shock

[0730] Repeat Example 12.13, except that the two pulses have a pulse width of 0.5 seconds and a time interval of 0.5 seconds.

[0731] Example 14.2 – Two-pulse electric shock

[0732] Repeat Example 14.1, except that each pulse is +0.0V to +2.0V.

[0733] Example 14.3 – Multiple-pulse electric shock

[0734] Repeat Example 12.13, except that a series of 10 pulses have a pulse width of 0.1 seconds and a time interval of 0.1 seconds between two pulses.

[0735] Example 14.4 – Multiple-pulse electric shock

[0736] Repeat Example 14.1, except that each pulse is +0.0V to +2.0V.

[0737] Example 14.5 – Single-stage incremental electric shock

[0738] Repeat Example 12.13, except that the potential is gradually increased from +0.0V to +1.0V over a period of 1 second.

[0739] Example 14.6 – Multi-stage escalating electric shock

[0740] Repeat Example 14.5, except that the increasing potential is repeated 5 times, with a time interval of 0.1 between the two increases.

[0741] Example 14.7 – Single-stage incremental electric shock

[0742] Repeat Example 12.13, except that the potential is gradually increased from +0.0V to +2.0V over a period of 2 seconds.

[0743] Example 14.8 – Multi-stage escalating electric shock

[0744] Repeat Example 14.7, except that the increasing potential is repeated 5 times, with a time interval of 0.1 between the two increases.

[0745] Adjusting the working electrode

[0746] Example 15.1 – Preparation of a glucose sensing system in serum

[0747] Example 10.2 was repeated to prepare an electrochemical cell for glucose sensing in serum. The working electrode 103 was one of the electrodes 1607 prepared in Example 8.4 (including a platinum nanoporous layer 1609) and did not include an electrolyte ion blocking layer.

[0748] Example 15.2 – Adjusting the working electrode (without electrolyte ion blocking layer)

[0749] In the electrochemical cell prepared in Example 15.1, a bias voltage of 0.4V was applied between the working electrode 103 and the reference electrode 106. Unlike Example 10.3, the current from the working electrode was measured continuously immediately after the bias voltage was applied. Figure 42A A graph showing the current distribution measured at the electrochemical cell is shown, where the working electrode 103 does not include the electrolyte ion barrier layer. See also Figure 42A At 10,000 seconds (approximately 3 hours), 20,000 seconds, and 30,000 seconds, the current still decreased at a significant rate. Figure 42B yes Figure 42A An enlarged view of the graph shows that the glucose stock solution prepared as in Example 9.1 was added only after the working electrode had been conditioned.

[0750] Example 15.3 – Fabrication of a working electrode with a PMMA electrolyte ion-blocking layer

[0751] PMMA (product number 445746) purchased from Sigma-Aldrich was dissolved in dimethylformamide (DMF) to provide a 2 wt% PMMA solution. Using a microsyringe, 0.2 μL of the PMMA solution was dropped onto the platinum nanoporous layer 1609 of one of the electrodes 1607 prepared in Example 8.4. As the solvent dried, a PMMA electrolyte ion blocking layer 505 was formed on the platinum nanoporous layer 1609.

[0752] Example 15.4 – Preparation of a glucose sensing system in serum

[0753] To prepare an electrochemical cell for sensing glucose in serum, Example 10.2 was repeated, except that the working electrode with a PMMA electrolyte ion-blocking layer prepared in Example 15.1 was used as working electrode 103.

[0754] Example 15.5 – Adjusting the working electrode

[0755] In the electrochemical cell prepared in Example 15.4, a bias voltage of 0.4V was applied between the working electrode 103 and the reference electrode 106. After the bias voltage was applied, the current from the working electrode was measured continuously immediately. Figure 43 A graph showing the current distribution measured from the electrochemical cell is shown, where the working electrode 103 includes an electrolyte ion blocking layer. The glucose stock solution prepared as in Example 9.1 was added only after the working electrode had been conditioned. Figure 43 The peaks in the text represent the stirring after each addition.

[0756] Example 15.6 – Comparison Adjustment Time

[0757] Figure 44 Covering Figure 42 (Example 15.2) and Figure 43 The current distribution diagram for Example 15.5 is shown. The current in Example 15.5 (including the electrolyte ion blocking layer) is fixed and stable for about 600 seconds, while the current in Example 15.2 (without the electrolyte ion blocking layer) decreases at a significant rate during the same time period.

[0758] Example 15.7 – Fabrication of a working electrode with a PHEMA layer

[0759] PHEMA (product number 529265) purchased from Sigma-Aldrich was dissolved in dimethylformamide (DMF) to provide a 2 wt% PHEMA solution. Using a microsyringe, 0.2 μL of the PHEMA solution was dropped onto the platinum nanoporous layer 1609 of one of the electrodes 1607 prepared in Example 8.4. As the solvent dried, a PHEMA electrolyte ion blocking layer 505 was formed on the platinum nanoporous layer 1609.

[0760] Example 15.8 – Fabrication of a working electrode with a PMMA-EG-PMMA layer

[0761] PMMA-EG-PMMA (product number 463183), purchased from Sigma-Aldrich, was dissolved in dimethylformamide (DMF) to provide a 2 wt% PMMA-EG-PMMA solution. Using a microsyringe, 0.2 μL of the PMMA-EG-PMMA solution was dropped onto the platinum nanoporous layer 1609 of one of the electrodes 1607 prepared in Example 8.4. As the solvent dried, a PMMA-EG-PMMA electrolyte ion blocking layer 505 was formed on the platinum nanoporous layer 1609.

[0762] Example 15.8 – Preparation and Regulation of a Glucose Sensing System in Serum

[0763] An electrochemical cell for sensing glucose in serum was prepared by repeating Example 15.4, except that the working electrode prepared in Examples 15.7 and 15.8 was used as working electrode 103. Furthermore, Example 15.5 was repeated for the prepared electrochemical cell.

[0764] Manufacturing CGM subcutaneous electrode units

[0765] Example 16.1 – Forming a conductive layer on a substrate

[0766] A 150 μm thick polyimide film was used as the substrate 503. A silver layer 1603 was printed on the polyimide film to provide silver conductive elements 110C, 110W, and 110R with a thickness of approximately 20 μm, shaped as follows. Figure 35 As shown. Subsequently, a conductive carbon layer 1605 was printed on the silver conductive elements 110C and 110W with a thickness of approximately 20 μm. No carbon layer was formed on the silver conductive element 110R.

[0767] Example 16.2 – Placing and Cutting the Insulating Layer

[0768] A 50 μm thick polyimide film is used as the insulating layer 707. The polyimide film is cut to specific dimensions for coverage. Figure 35 The intermediate product is used to expose the terminal portion 705. The polyimide film is punctured to provide three openings for exposing the areas of the working electrode, reference electrode, and counter electrode. Subsequently, the pre-cut polyimide is placed... Figure 35 The intermediate product is used to make the adhesive layer contact the polyimide substrate 503 for providing Figure 36 The intermediate product. Subsequently, the polyimide substrate 503 and polyimide insulating layer 707 outside the conductive element are cut to provide Figure 37 Intermediate products.

[0769] Example 16.3 – Formation of Clustered Nanoporous Layers

[0770] The cluster colloid obtained in Example 5.1 was diluted with purified water to 60 mg / ml. Using a microsyringe, 0.2 μL of the diluted cluster colloid was dropped onto the carbon layer 1605 exposed through an opening in the working electrode 501 of the intermediate product prepared in Example 16.2. The cluster colloid dropped onto the carbon layer 1605 was dried to provide a clustered nanoporous layer 117, thereby producing Figure 38A Intermediate products.

[0771] Example 16.4 – Formation of an electrolyte ion blocking layer

[0772] PMMA (product number 445746) purchased from Sigma-Aldrich was dissolved in dimethylformamide (DMF) to provide a 2 wt% PMMA solution. Using a microsyringe, 0.2 μL of the PMMA solution was dropped onto the nanoporous layer 117 of the intermediate prepared in Example 16.3. As the solvent dried, a PMMA electrolyte ion barrier layer 505 was formed on the nanoporous layer 117.

[0773] Example 16.5 – Formation of a biocompatible layer

[0774] A biocompatible layer (pHEMA) is formed on the electrolyte ion barrier layer 505, such as Figure 38B As shown, thus producing Figure 33 The non-enzymatic CGM electrode unit.

[0775] Example 16.6 – Formation of a biocompatible layer

[0776] pHEMA (product number 192066), purchased from Sigma-Aldrich, was dissolved in dimethyl sulfoxide (DMSO) to provide a 0.5 wt% pHEMA solution. Using a microsyringe, 1.0 μL of the pHEMA solution was dropped onto the electrolyte ion barrier layer 505 of the intermediate prepared in Example 16.4. When the solvent dried, as... Figure 38B The pHEMA biocompatibility layer 507 is formed as shown, thereby producing Figure 33 The non-enzymatic CGM electrode unit 701.

[0777] CGM Animal Testing

[0778] Example 17.1 – Preparing for CGM Animal Testing

[0779] The non-enzymatic CGM electrode unit prepared in Example 16.6 was subcutaneously inserted into a rat so that electrodes 103, 105, and 106 were in contact with the rat's tissue fluid. The CGM electrode unit 701 was connected to an UXN regulator developed by UXN Co., Ltd. (the applicant of this application) and Seoul National University Hospital. Figure 45A This is a photo of the UXN voltage regulator. Figure 45B This illustrates the connection of the CGM electrode unit 701 to... Figure 45A A photo of the UXN regulator. Figure 45C This is a photograph showing the UXN voltage regulator with its casing. The UXN voltage regulator includes a wireless module for wireless communication with a computer, and the UXN voltage regulator can be wirelessly controlled by the computer. A glucose solution was prepared for injection into the veins of rats to induce changes in glucose levels in the rat's blood and tissue fluid.

[0780] Example 17.2 – Continuous monitoring of glucose levels in rats

[0781] The CGM electrode unit 701 was maintained subcutaneously for 5 consecutive days. On the first day, rats were injected with glucose solution twice. For the following days, glucose solution was injected once daily. The current from the CGM electrode unit 701 was measured using a UXN regulator approximately 1.5 hours after the first injection each day. Additionally, every 2–5 minutes during this approximately 1.5-hour period, a small amount of blood was collected from the rat's tail and applied to the Roche Accu-Tex database. The blood glucose meter's test strips provide the concentration of glucose in the blood.

[0782] Example 17.3 – Mapping CGM measurement data and blood glucose in rats

[0783] Figure 46 The current from the CGM electrode module, measured by the UXN regulator of Example 17.2, is shown in blue. Figure 46 The red dot in the image indicates the location from Roche Accu The blood glucose concentration obtained from the blood glucose meter. Considering the approximately 10-minute time lag between glucose levels in tissue fluid and blood glucose levels, the data was calibrated by timely movement of the blue signal relative to the red dot. It should be understood that the spikes in the blue signal primarily originate from the rat's body movements during the measurement. Based on the photograph in Figure 45, using the Roche Accu... There appears to be a strong correlation between the blood glucose concentration measured by the blood glucose meter and the CGM monitored using the non-enzymatic CGM electrode unit 701 prepared in Example 16.6.

[0784] Example 17.4 – Clark Error Grid Analysis

[0785] Figure 47 Based on Figure 46 The photograph shows the measurement data from the Clarke error grid of the non-enzymatic CGM electrode unit 701 prepared in Example 16.6. The reference sensor used for this Clarke error grid analysis is a Roche sensor. Blood glucose meter. The grid has five zones. Zone A includes values ​​within the 20% reference sensor range; Zone B includes values ​​outside the 20% range of Zone A but that do not lead to inappropriate treatment; Zone C includes values ​​that may lead to unnecessary treatment; Zone D includes values ​​indicating a potential risk of failure in detecting hypoglycemia or hyperglycemia; and Zone E includes values ​​that could confuse treatment of hypoglycemia with hyperglycemia (and vice versa). As summarized in the table below the grid, the analysis shows that over 91% of the points are in Zones A and B.

[0786] Combination of features

[0787] This disclosure provides extensive discussion and information regarding numerous features of nanoporous structures and / or glucose sensing technologies. The intent of this disclosure is to provide as many devices, systems, and methods as possible related to those features. Two or more features disclosed above may be combined to form devices, systems, or methods, even if a particular combination is not proposed in this disclosure. Furthermore, the intent of this disclosure is to seek claims relating to many of the features disclosed herein. Some of those features are set forth in the form of the claims hereinafter. Many claims are set forth in dependent form, referring to one or more other claims. The applicant notes that some claims referring to multiple claims may cover combinations of features that conflict with each other (hereinafter referred to as “inappropriate combinations”). However, the applicant recognizes that such claims may still cover one or more combinations of features that do not conflict with each other (hereinafter referred to as “appropriate combinations”). By setting forth claims that can cover both appropriate and inappropriate combinations, the applicant demonstrates its or the inventor’s ownership of appropriate combinations and intends to provide specific support for future claims to appropriate combinations of these appropriate combinations.

Claims

1. A nanoporous layer comprising: The deposit comprises an irregularly shaped matrix of numerous nanoparticles, which are generally elliptical or spherical in shape and have lengths between 2 nm and 5 nm. The adjacent irregular-shaped main bodies are adjacent to each other, while creating unoccupied spaces between them. Adjacent irregular-shaped entities further connect to other irregular-shaped entities, forming a three-dimensional interconnected network of irregular-shaped entities. The unoccupied spaces between adjacent irregularly shaped entities are connected to other unoccupied spaces formed by other irregularly shaped entities, forming a three-dimensional interconnection network of irregularly shaped spaces. This three-dimensional interconnection network of irregularly shaped spaces is provided outside the three-dimensional interconnection network of the irregularly shaped entities. in, Within the three-dimensional interconnected network of the irregularly shaped body, at least a portion of the nanoparticles are adjacent to each other without intermediate nanoparticles and separated from each other by interparticle nanopores. The nanoporous layer is contained in the interparticle nanopores within the three-dimensional interconnection network of the irregularly shaped body, and also in the three-dimensional interconnection network of the irregularly shaped space outside the three-dimensional interconnection network of the irregularly shaped body.

2. The nanoporous layer of claim 1, wherein at least a portion of the interparticle nanopores within the three-dimensional interconnection network of the irregularly shaped body comprises gaps with a size ranging from 0.5 nm to 3 nm.

3. The nanoporous layer of claim 1, wherein at least a portion of the irregular space of the three-dimensional interconnection network comprises gaps with a size range between 100 nm and 500 nm.

4. The nanoporous layer of claim 1, wherein the interparticle nanopores are typically distributed throughout the three-dimensional interconnection network of the irregularly shaped body, and wherein the unoccupied space of the three-dimensional interconnection network of the irregularly shaped space is typically distributed throughout the nanoporous layer.

5. The nanoporous layer of claim 1, wherein the interparticle nanopores are substantially interconnected within and further connected to the three-dimensional interconnection network of the irregular body.

6. The nanoporous layer of claim 1, wherein the nanoporous layer does not contain organic molecules or, if present, contains less than 0.5 parts by weight of organic molecules based on 100 parts by weight of the deposit.

7. The nanoporous layer of claim 1, wherein the nanoporous layer has a roughness coefficient between 100 and 2500.

8. A glucose sensing electrode, comprising: Substrates including conductive surfaces; and The nanoporous layer formed on the conductive surface according to any one of claims 1 to 7.

9. The glucose sensing electrode of claim 8, wherein the glucose sensing electrode does not contain a glucose-specific enzyme.

10. The glucose sensing electrode of claim 8, wherein the nanoporous layer does not contain organic molecules or contains less than 0.5 parts by weight of organic molecules based on 100 parts by weight of the deposit.

11. The glucose sensing electrode of claim 8, wherein the substrate comprises a conductive metal layer and a conductive carbon layer formed on the conductive metal layer, wherein the substrate comprises a conductive or semi-conductive material providing the conductive surface.

12. The glucose sensing electrode according to claim 8, When a bias voltage of 0.2-0.45V is applied between the glucose sensing electrode and the reference electrode in contact with a glucose-containing liquid, the glucose sensing electrode is configured to oxidize the glucose in the nanoporous layer and to generate a current. This current is the sum of the glucose oxidation current generated solely by glucose oxidation and the background current generated by other electrochemical interactions between the glucose-containing liquid and the glucose sensing electrode. When the glucose-containing liquid contains glucose at a concentration of 4-20 mM (72-360 mg / dL), the glucose oxidation current is above 10 nA / mMcm under steady state. 2 At the level of.

13. The glucose sensing electrode of claim 8, further comprising: An electrolyte ion barrier layer formed on the nanoporous layer; and a biocompatible layer formed on the electrolyte ion barrier layer, wherein, upon contact with a substance containing glucose, Na+, or sodium-containing electrolytes, the electrolyte is protected against ion buildup. + K + Ca 2+ Cl - PO4 3- and CO3 2- In the case of a liquid, the electrolyte ion blocking layer is configured to suppress the Na+ contained in the liquid. + K + Ca 2+ Cl - PO4 3- and CO3 2- Diffusion is made into the nanoporous layer to create a Na+ layer between the top and bottom of the electrolyte ion barrier layer. + K + Ca 2+ Cl - PO4 3- and CO3 2- The general discontinuity of the combined concentration.

14. The glucose sensing electrode of claim 13, wherein the combined concentration below the electrolyte ion blocking layer is greater than 0% and less than 10% of the combined concentration above the electrolyte ion blocking layer.

15. The glucose sensing electrode of claim 13, wherein the electrolyte ion blocking layer comprises a porous hydrophobic polymer layer configured to restrict Na+ ions. + K + Ca 2+ Cl - PO4 3- and CO3 2- Migration through it, However, it does not restrict the migration of glucose molecules through it.

16. The glucose sensing electrode of claim 13, wherein the electrolyte ion blocking layer comprises at least one selected from the group consisting of: poly(methyl methacrylate) (PMMA), poly(hydroxyethyl methacrylate) (PHEMA), and poly(methyl methacrylate-co-ethylene glycol dimethacrylate) (PMMA-EG-PMMA).

17. A glucose sensing device, comprising: Single entity; A first electrode, comprising the glucose sensing electrode according to any one of claims 9 to 16 and formed on the single body; and A second electrode is formed on the single body and configured to contact the liquid when the first electrode contacts the liquid.

18. The apparatus of claim 17, wherein the glucose sensing device does not include a glucose-specific enzyme.

19. The device of claim 17, wherein the nanoparticles of the nanoporous layer are made of at least one selected from the group consisting of platinum (Pt), gold (Au), palladium (Pd), rhodium (Rh), titanium (Ti), ruthenium (Ru), tin (Sn), nickel (Ni), copper (Cu), indium (In), thallium (Tl), zirconium (Zr), iridium (Ir), and one or more oxides of the aforementioned metals, wherein the first electrode does not include a biocompatible layer configured to suppress immune rejection.

20. The device of claim 17, wherein the nanoparticles comprise at least one of platinum (Pt) and gold (Au), wherein the first electrode comprises a biocompatible layer configured to suppress immune rejection.

21. A non-enzymatic glucose sensing method, the method comprising: Provide the apparatus according to any one of claims 18 to 20; When the test fluid comes into contact with both the first electrode and the second electrode, a bias voltage is applied between the first electrode and the second electrode, which causes the oxidation of glucose contained in the test fluid in the glucose sensing electrode. and Measure the current flowing from the first electrode; and process the measured current, with or without additional data, to provide a glucose level corresponding to the glucose contained in the test fluid.

22. A method for preparing a colloid, the method comprising: A liquid composition comprising a surfactant and a metal ion is provided, wherein the surfactant is in a reverse micelle phase comprising a plurality of hydrophilic spaces; Adding a reducing agent to the liquid composition to reduce at least a portion of the metal ions to form nanoparticles provides a first colloid, wherein at least a portion of the nanoparticles are located within at least some of the plurality of hydrophilic spaces, wherein no potential is applied for the reduction of at least a portion of the metal ions; as well as The surfactant is removed from the first colloid to form a second colloid comprising a plurality of irregularly shaped masses dispersed in the liquid, wherein each irregularly shaped mass contains a cluster of nanoparticles having a generally elliptical or spherical shape and a length in the range of 2 nm to 5 nm. The irregularly shaped body comprises a first cluster and a second cluster discretely dispersed in the liquid, wherein the first cluster and the second cluster each have a length between 50 nm and 300 nm.

23. The method of claim 22, wherein the first cluster comprises a first nanoparticle and a second nanoparticle, each having a generally elliptical or spherical shape and a length between 2 nm and 5 nm, wherein within the first cluster, the first nanoparticle and the second nanoparticle are adjacent to each other without any intermediate nanoparticles between them, and are separated from each other by a first interparticle gap with a size ranging from 0.5 nm to 3 nm.

24. The method of claim 22, wherein some of the surfactant molecules are bound to the nanoparticles in the first colloid, wherein removing the surfactant further comprises adding an acid or base to the first colloid to separate at least a portion of the molecules from the nanoparticles.

25. The method of claim 22, wherein, After removing the surfactant, the method further includes adjusting the concentration of the nanoparticles in the second colloid to provide a colloidal composition such that the amount of the nanoparticles contained in the colloidal composition is between 0.01 wt% and 2 wt% based on the total weight of the colloidal composition.

26. A method for preparing a nanoporous layer, the method comprising: Perform the method of any one of claims 22 to 25 to provide the second colloid; Subsequently, the concentration of the nanoparticles in the second colloid is adjusted to provide a colloidal composition; The colloidal composition is dispensed onto a substrate; as well as The dispensed colloidal composition is subjected to drying to form a nanoporous layer.

27. The method of claim 26, wherein by adjusting the concentration of the nanoparticles, the amount of the nanoparticles contained in the colloidal composition is between 0.01 wt% and 2 wt% based on the total weight of the colloidal composition.

28. The method of claim 26, The dispensed colloidal composition is subjected to drying to deposit irregularly shaped entities contained in the dispensed colloidal composition onto the substrate, such that adjacent irregularly shaped entities are adjacent to each other, while unoccupied spaces are formed between them. Adjacent irregular-shaped entities are further connected to other irregular-shaped entities, forming a three-dimensional interconnected network of irregular-shaped entities. The unoccupied space is connected to other unoccupied spaces formed by other irregularly shaped entities to form a three-dimensional interconnection network of irregularly shaped spaces, which is provided outside the three-dimensional interconnection network of the irregularly shaped entities.

29. The method of claim 28, wherein the three-dimensional interconnection network of the irregularly shaped body comprises a plurality of nanoparticles derived from the irregularly shaped body of the colloidal composition and having a generally elliptical or spherical shape, the nanoparticles having a length in the range of 2 nm to 5 nm.

30. The method of claim 28 or 29, wherein, Within the three-dimensional interconnected network of the irregularly shaped body, at least a portion of the nanoparticles are adjacent to each other without intermediate nanoparticles and separated from each other by interparticle nanopores.

31. The method of claim 30, wherein the nanoporous layer comprises the interparticle nanopores within the three-dimensional interconnection network of the irregularly shaped body, and further comprises a three-dimensional interconnection network in an irregularly shaped space outside the three-dimensional interconnection network of the irregularly shaped body.

32. The method of claim 30, wherein at least a portion of the interparticle nanopores within the three-dimensional interconnected network of the irregularly shaped body comprises gaps with sizes ranging from 0.5 nm to 3 nm.

33. The method of claim 28 or 29, wherein at least a portion of the irregular space of the three-dimensional interconnection network comprises gaps with dimensions ranging from 100 nm to 500 nm.

34. The method of claim 31, wherein at least a portion of the interparticle nanopores within the three-dimensional interconnected network of the irregularly shaped body comprises gaps with sizes ranging from 0.5 nm to 3 nm.

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